In "The Wrestler," which was the last in-competition movie screened at the 65th edition of the storied festival, Rourke plays a fighter with heart problems who tries to reconnect with his estranged daughter while feeling the dangerous allure of a return to the ring.
"I need to thank Mickey Rourke, for opening up his heart and soul for the camera, for trusting me and giving me the honor of reminding the world what a great talent he is," Aronofsky said after receiving the award.
German filmmaker Wim Wenders, who headed the Venice jury, announced the winning movie and played on the lead character's heart problems to praise Rourke's work, which he called "a truly heartbreaking performance, in the very sense of the word."
Rourke, who joined Aronofsky on the stage, thanked the jury "for making the right decision" and congratulated the director of "The Fountain" and "Requiem for a Dream" for choosing to make "The Wrestler."
"Darren could make other kind of movies if he wanted to, make a lot of money in the United States," Rourke said. "I love and respect him because he doesn't compromise and he wants to make movies that are not that expensive, that have a lot of integrity."
Aronofsky dedicated the award "to all the wrestlers who we met along the way, who are working for 200 bucks a night ... who just want to entertain and are willing to sacrifice their bodies and their souls for it."
Russia's Aleksey German Jr. won the award for best director for "Bumaznyj soldat (Paper Soldier)," a story set in the early days of the space program in the Soviet Union.
The emotional director thanked his cast and especially one of the producers, who sold his apartment to pay for the film.
The festival's jury awarded a special award to German filmmaker Werner Schroeter, who was competing with "Nuit de chien."
France's Dominique Blanc won best actress for playing a jealousy-struck woman in "L'Autre (The Other One)," directed by Patrick Mario Bernard and Pierre Trividic.
Italian Silvio Orlando was crowned best actor for his role in Pupi Avati's "Il Papa' di Giovanna," (Giovanna's Father), about a father's relationship with his troubled adult daughter.
Most of the winners heaped praise on jury head Wenders, whose credits include "Paris, Texas" and "Wings of Desire."
"We grew up with him," Orlando said. "In high school we would stop studying ... to see his movies."
But Wenders ended the ceremony on a surprise note, taking the microphone after the awards were handed out to criticize the festival's rules, which dictate that the best performance awards must go to different films than those that take the top prizes.
"That creates conflict between the heart and the mind" of the jurors, he said. "We recommend that you reconsider."