Michele Scarponi, who won the Giro d'Italia in 2011, died after being hit by a van while training on Saturday. He was 37.
Scarponi was training near his home of Filottrano, near Ancona, when he was hit by a van at a crossroad. He leaves behind a wife and twin sons.
According to initial reports the Astana cyclist died on the spot and was unable to be revived by emergency services which arrived promptly.
Team Astana called it "a tragedy too big to be written" in a statement.
"We left a great champion and a special guy, always smiling in every situation, he was a real mile stone and a landmark for everyone in the Astana Pro Team," it added.
Scarponi was awarded the 2011 Giro trophy after Alberto Contador was stripped of the title because of doping.
He had been named as Astana's leader for the upcoming race, which starts in less than two weeks, after Fabio Aru pulled out with a knee injury.
Scarponi had returned home after finishing fourth in the Tour of the Alps on Friday in Trento. He won the first stage on Monday.
The Italian was one of the most liked riders on the circuit, with his sense of humor and jovial disposition.
"Yesterday he was racing. He came up to me. Michele was smiling, as ever," Italy team coach Davide Cassani wrote on Twitter. "He was happy for (Monday's) win. He was talking about the Giro. And now I'm here crying for him. Oh my God."