The move extended a prolonged legal battle that has become a cause celebre in the United States and raised a host of questions about how the next phase of Italian justice would play out.
Knox, now a 25-year-old University of Washington student in Seattle, called the decision by the Rome-based Court of Cassation "painful" but said she was confident that she would be exonerated.
The American left Italy a free woman after her 2011 acquittal - but only after serving nearly four years of a 26-year prison sentence from a lower court that convicted her of murdering Meredith Kercher. The 21-year-old British exchange student's body was found in November 2007 in a pool of blood in the bedroom of a rented house the two shared in the Italian university town of Perugia. Her throat had been slit.
Raffaele Sollecito, Knox's Italian boyfriend at the time, was also convicted, sentenced and later acquitted.
It could be months before a date is set for a fresh appeals court trial for Knox and Sollecito in Florence, which was chosen because Perugia has only one appellate court.
Italian law cannot compel Knox to return for the new trial and one of her lawyers, Carlo Dalla Vedova, said she had no plans to do so.
"She thought that the nightmare was over," Dalla Vedova told reporters on the steps of the courthouse. "(But) she's ready to fight."
He spoke minutes after relaying the top court's decision to Knox by phone shortly after 2 a.m. local time in Seattle.
Another Knox defender, Luciano Ghirga, was gearing up psychologically for his client's third trial. Ghirga said he told Knox: "You have always been our strength. We rose up again after the first-level convictions. We'll have the same resoluteness, the same energy" in the new trial.
Still, it was a tough blow for the former exchange student, whose parents have had to mortgage both their homes to raise funds for her lengthy, expensive defense.
"It was painful to receive the news that the Italian Supreme Court decided to send my case back for revision when the prosecution's theory of my involvement in Meredith's murder has been repeatedly revealed to be completely unfounded and unfair," Knox said in a statement.
Knox said the matter must now be examined by "an objective investigation and a capable prosecution."
"No matter what happens, my family and I will face this continuing legal battle as we always have, confident in the truth and with our heads held high in the face of wrongful accusations and unreasonable adversity," Knox said.
Prosecutors alleged that Kercher was the victim of a drug-fueled sex game gone awry. Knox and Sollecito denied wrongdoing and said they weren't even in the apartment that night, although they acknowledged they had smoked marijuana and their memories were clouded.
An Ivory Coast man, Rudy Guede, was convicted of the Kercher slaying in a separate proceeding and is serving a 16-year sentence.
Sollecito, whose 29th birthday was Tuesday, sounded shaken when a reporter reached him by phone.
"Now, I can't say anything," said the Italian, who has been studying computer science in the northern city of Verona after finishing up an earlier degree while in prison.
A local Italian news report quoted Sollecito's current girlfriend as saying he and Knox spoke by phone after the judicial setback and described him as being psychologically destroyed.
His lawyer, Luca Maori, said neither Sollecito nor Knox ran any danger of being arrested.
"It's not as if the lower-court convictions are revived," he said, noting that the Cassation Court didn't pronounce "whether the two were innocent or guilty. "
For those familiar by the U.S. legal principle of "double jeopardy" - by which no one who is acquitted of a crime can be tried again for it - the idea that Italian justice system allows prosecutors to appeal acquittals is hard to absorb.
Knox attorney Dalla Vedova dismissed the "double jeopardy" concern, insisting the high court ruling Tuesday hadn't decided anything about the defendants' guilt or innocence, but merely ordered a fresh appeals trial.
Knox still planned to talk with celebrity interviewer Diane Sawyer in a prime-time special to be broadcast April 30 to promote her new book "Waiting to Be Heard," according to ABC News.
Dalla Vedova said Knox wouldn't come to Italy but would follow the case from home. He said he didn't think the new appeals trial would begin before early 2014 and no date would be set for it until after the top court issues a written explanation of its decision, due in the next 90 days.
Whether Knox ever returns to Italy to serve more prison time depends on a string of ifs and unknowns.
"Questions of extradition are not in the legal landscape at this point," another Knox attorney, Theodore Simon, said on NBC TV.
If she is convicted by the Florence court, Knox could appeal that verdict to the Cassation Court, since Italy's judicial system allows for two levels of appeals - by prosecutors and the defense alike. Should that appeal fail, Italy could seek her extradition from the United States.
Whether Italy actually requests extradition will be a political decision made by a future Italian government.
In the past, Italian governments on both the left and the right refused Italian prosecutors' request to seek extradition for the trial of 26 Americans accused in the kidnapping of an Egyptian cleric in Milan under the CIA's extraordinary rendition program. All 26 were tried in absentia, convicted and received sentences ranging from seven to nine years. Italy's new government will decide if it wants seek their extradition to serve the sentences.
If Knox is convicted and Italy requests her extradition, it would be up to U.S. authorities to decide whether they will send Knox to Italy. Dalla Vedova noted that U.S. authorities would carefully study all the case's documentation to decide whether she had received fair trials.
U.S. and Italian authorities could also come to a deal that would keep Knox in the U.S.
Years ago, the United States extradited an Italian woman convicted in a domestic U.S. terrorism case after reaching a deal that she would serve the rest of her sentence in her homeland. Italian authorities, however, released her from prison not long after she arrived home, citing medical reasons.
The appeals court that acquitted Knox and Sollecito had criticized virtually the entire case mounted by prosecutors, and especially the forensic evidence which helped clinch their 2009 convictions. The appellate court noted that the murder weapon was never found, said that DNA tests were faulty and that prosecutors provided no murder motive.
In arguing for the acquittals to be overturned, the prosecutor described the Perugia appellate court as being too dismissive about whether DNA tests were reliable on a knife prosecutors allege could have been the one used to slash Kercher's throat and DNA traces on a bra belonging to the victim, as well as tests done on blood stains in the bedroom and bathroom.
Whether that argument swayed the top court was unclear, said Dalla Vedova. Sollecito's attorney, Giulia Bongiorno, said the appeals court might have been "too generous" in ruling that the pair did not commit the crime, but was confident that Sollecito's innocence would be affirmed.
The court on Tuesday also upheld a slander conviction against Knox. During a 14-hour police interrogation, Knox had accused a local Perugia pub owner of carrying out the killing. The man was held for two weeks based on her allegations, but was then released for lack of evidence.
Her defense lawyers say Knox felt pressured by police to name a suspect so her own interrogation could end.
Because of the time she served in prison before the acquittal, Knox didn't have to serve the three-year sentence for the slander conviction. The court on Tuesday also ordered Knox to pay 4,000 euros ($5,500) to the man, as well as the cost of the lost appeal.
It was not known why the top court concluded the appellate court had erred in acquitting Knox and Sollecito and won't be until the Cassation judges issue their written ruling.
But Prosecutor General Luigi Riello, who urged the Cassation judges to overturn the acquittals, said he thought it could be significant that Knox's slander conviction was upheld. If the Cassation judges think "there is a link" between Knox's reason for fingering the pub owner and the murder, it could bolster prosecutors in the new Florence trial, he said.
In her statement, Knox took the Perugia prosecutors to task, saying they "must be made to answer" for the discrepancies in the case. She also said "my heart goes out to" Kercher's family.
The Kercher family's attorney, Francesco Maresca, called Tuesday's ruling "what we wanted" and relayed a message quoting the late woman's sister, Stephanie.
"To understand the truth about what happened that night is all we can do for her now," the family's message said.
AP writer Colleen Barry in Milan contributed to this report.