The home team's best chances fell to Pablo Osvaldo and Mario Balotelli in a dominant spell before halftime but the new attacking duo could not find a way past Fernando Muslera on the goalkeeper's return to Rome.
Alvaro Pereira was sent off with nine minutes left on the clock but Uruguay, who thrashed Chile 4-0 in a World Cup qualifier on Friday, held on.
"This really is a significant win," coach Oscar Tabarez said. "To beat an historic rival such as Italy away has finished off a spectacular year for us.
"Italy were good with the ball but our young players have given their all and shown they are up to the job. Our defence was strong and that's why they couldn't break through."
Uruguay took the lead on a cold night when Martin Caceres's right-wing cross reached Fernandez near the penalty spot and the striker, playing in place of Friday's four-goal hero Luis Suarez, sidefooted the ball into the corner.
Balotelli, who scored his first goal for Italy in Friday's friendly win in Poland, almost doubled his account on 17 minutes when he warmed Muslera's fingers with a blistering strike after finding space 25 metres out.
Two minutes later Osvaldo was left free at the back post but the Roma striker's header drifted wide.
Osvaldo came close to equalising 10 minutes before halftime but watched agonisingly as his textbook near-post header from Andrea Pirlo's corner bounced the wrong side of the far post.
With Italy controlling the game, Balotelli shot weakly at Muslera after twisting in the box before he headed Federico Balzaretti's left-wing cross over the bar just before half-time.
Former Lazio goalkeeper Muslera had to pull off a fine one-handed save at his near post from substitute Simone Pepe 10 minutes after the break before Alessandro Matri volleyed a metre wide from the edge of the box.
Muslera was again in action as he turned Balzaretti's close-range effort round the post but despite losing Pereira for a second caution, Uruguay hung on for victory.
"They are obviously full of confidence, a real team that knows how to manage a game," said Prandelli. "But I thought we played well against a team that are South American champions and were fourth in the World Cup.
"We took it seriously right up to the 95th minute and the players were very disappointed to lose. Tonight I can say we have a real team."