Late goals from Paris St Germain striker Edinson Cavani and Cristian Stuani handed the two-time FIFA World Cup winners a dramatic win at the Centenario Stadium as Colombia's hopes of sealing their passage were put on hold.
The Colombians had needed only a point to be sure of clinching their first FIFA World Cup qualification since 1998 and for long periods looked more than capable of claiming the result they needed.
But Uruguay - who are now firm favourites to achieve at least a play-off spot - broke the deadlock 13 minutes from time.
A superb curling cross from Maxi Pereira was met by Cavani, who guided his header expertly past Colombian keeper David Ospina to make it 1-0.
Moments later Gaston Ramirez carved out some space inside the area and cut back to find Cristian Stuani who coolly tucked the ball away to make it 2-0.
It was a third consecutive victory for Uruguay in the qualifying tournament and left them level on 22 points with fourth placed Ecuador, who drew 1-1 with Bolivia earlier on Tuesday.
Second-placed Colombia remain firmly on course to qualify however, requiring only a point from their final two fixtures to reach Brazil.