Mexico-based midfielder Edison Mendez struck the winner with a low drive from the edge of the penalty area in the 77th minute on a wet afternoon at the Atahualpa stadium.
Brazil began the day top of the 10-nation group with 20 points from 10 games, one point ahead of Argentina who were at home to Venezuela later on Wednesday. Ecuador moved on to 16 points.
The world champions, whose players arrived in Ecuador from Europe less than 48 horus before kickoff, suffered from a combination of fatigue and the high altitude of Quito at 2,800 metres above sea level.
But they still had the better of the game until Mendez struck to give Ecuador a repeat of their historic win over Brazil in the 2002 World Cup qualifiers at the same venue.
The visitors could have enjoyed a halftime lead if Ronaldo and Kaka had been on the same wavelength.
Kaka wasted an excellent chance in the eighth minute when he failed to spot Ronaldo in a good position.
Three minutes later, Ronaldo broke clear on the left and shot across the face of the goal with Kaka waiting unmarked in the penalty area.
Another chance went begging went Kaka shot straight at goalkeeper Edwin Villafuerte instead of crossing to Ronaldo, who gesticulated angrily.
Kaka also sidefooted wide from Cafu's low cross and shot into the side-netting after combining with Ronaldinho.
Agustin Delgado had Ecuador's best first-half effort with a close-range header which was brilliantly stopped by Dida after Mendez crossed from the right.
Brazil continued to dominate after the break and Ronaldinho had a shot tipped onto the crossbar from Villafuerte after his own free kick went into the wall and rebounded straight to him.
Villafuerte turned away a Kleberson drive and Kaka was also close.
But, as Ecuador began to create more chances, Renato nearly turned a Salas cross into his own net, Dida saving his blushes, and Delgado headed over with the goal at his mercy.
The winner came when Delgado, with his back to goal, played the ball out to Mendez whose firm right-foot shot appeared to take a deflection onto its way into the net.
Ronaldo completed a poor performance by side-footing wide from close range with three minutes to play.
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