The game was a "friendly" exhibition match. Clint Dempsey scored the only goal of the match in the 55th minute.
Dempsey took a short pass from Jozy Altidore following Michael Bradley's cross and put a right-footed shot from just inside the penalty area just past an outstretched hand of goalkeeper Gianluigi Buffon.
Although just an exhibition, it was a rare U.S. victory over a world power.
It was the 25th goal in 83 international appearances for Dempsey, a veteran of two World Cups and one of the players counted on to lead the Americans in qualifying for the 2014 tournament.
"Initially, I was going to stay where I was and have him lay it back to me," Dempsey said. "I saw the guy was covering. So I tried to move to my right to try get open so he could play me the ball. He did a great job of holding it up, playing me, and I just tried to hit it low and hard, far post, and it went in.
The U.S. was eliminated in the first round of the 2006 World Cup and in the second round in the 2010 Cup, beaten both times by Ghana.
The loss was particularly deflating for eighth-ranked Italy, a four-time World Cup champion preparing for the start of the European Championship in June. The No. 31 Americans, who are getting ready for their opening World Cup qualifier in June, had been 0-7-3 against Italy and had been outscored 32-4.
Italy dominated for stretches but the American defense held and the Azzurri were called for offsides nine times.
Italy lost in Genoa for the first time since 1924.
The Associated Press contributed to this report.