"The day after the election, some people, without logic or reason, called the glorious election a lie," state TV quoted Khamenei as saying.
He said questioning the election was "the biggest crime."
Opposition leader Mir Hossein Mousavi has said President Mahmoud Ahmadinejad stole the June 12 election from him through massive vote fraud. Hundreds of thousands of protesters poured into the streets in the days after the vote, prompting the government to stage a violent crackdown.
Although the street protests died down months ago, Mousavi and other leading opposition figures, including fellow presidential candidate Mahdi Karroubi and former President Mohammad Khatami, have refused to silence their protests.
Khamenei said he sent private messages to those who continue to question the election telling them they may not be able to control the future direction of events - an implied threat of additional government action if they fail to cease their activities. He did not name specific individuals.
The supreme leader has supported Ahmadinejad throughout the election crisis, including during the subsequent crackdown that resulted in hundreds of arrests and multiple deaths. The government has confirmed 30 people were killed, while the opposition says the 72 died.
The simmering post-election unrest has received less international attention in recent weeks, overshadowed by speculation about whether Iran will accept a U.N.-drafted plan to ship most of its low-enriched uranium to Russia for further enrichment.
Iran will deliver its response to the U.N. on Thursday, almost a week after the West had hoped to secure Tehran's approval, according to the semiofficial Mehr news agency. Iran needs the fuel for a research reactor that makes medical isotopes.
But it appears Iran wants to modify the deal to send its uranium stockpile out of the country in multiple batches rather than the big single shipment envisioned by the agreement - a change opposed by the West.
"Should we accept the option of sending uranium in return for fuel, we need to ship out a small part of it," the official Islamic Republic News Agency quoted lawmaker Hossein Ebrahimi as saying Wednesday.
"Should the other party remain committed to its obligations, this gradual trend will continue. We should not empty our uranium storehouses," said Ebrahimi.
Western powers say it's critical for Iran to send out at least 70 percent of its uranium store in one load as envisioned by the draft agreement to eliminate - at least temporarily - its options to make a nuclear weapon. Iran insists its nuclear program is peaceful.
The draft U.N. plan, which was formalized last week during talks in Vienna between Iran, Russia, France and the U.S., calls for Iran to send 2,420 pounds (1,100 kilograms) of low-enriched uranium to Russia in one batch by the end of the year.
After further enrichment in Russia, France would covert the uranium into fuel rods for return to Iran for use in the reactor in Tehran.
Significantly smaller batches or gradual shipments by Iran would leave the country with enough uranium to potentially produce a nuclear weapon.
A sum of 2,205 pounds (1,000 kilograms) is the commonly accepted amount of low-enriched uranium needed to produce weapons-grade uranium for a single nuclear warhead.