The 35-year-old American athlete had been in intensive care with severe brain damage and abdominal injuries at Cesena's Bufalini Hospital since Wednesday.
Hayden, who recently raced in nearby Imola, was struck by a Peugeot while training in Misano Adriatico on Wednesday.
Hailing from Kentucky, Hayden was the 2006 MotoGP champion, narrowly beating Valentino Rossi to take out the title for Honda.
Tributes have flowed across the world for the beloved athlete.
“Dear Nicky, you will be missed. May God hold you high, you are forever in our hearts. My thoughts and prayers to you and your loved ones,” Formula One’s Lewis Hamilton tweeted.
Italian judicial authorities have opened an investigation into the incident and questioned the 30-year-old driver of the car.
Hayden’s death is the latest in a string of incidents in which high-profile athletes have been injured or killed on European roads.
Last month, Italian cycling champion Michele Scarponi was killed while training nearby his home in Filottrano, Le Marche, when a van reportedly failed to see the 37-year-old former Giro d’Italia champion at an intersection.
Two weeks ago, British cyclist Chris Froome was involved in a hit-and-run incident near his home in Monaco, claiming he was “rammed on purpose”.
The three-time Tour de France champion escaped unharmed, but his bike was written off.
