Goals by Felipe Caicedo and Fidel Martinez gave the Andean side, who began with a shock 2-0 win over Argentina in Buenos Aires last month, the three points in Quito after Edinson Cavani had scored an equaliser for Uruguay.
The win was at altitude but Ecuador were missing four leading players including the England-based pair of Valencias, Antonio and Enner, while Uruguay, still without Luis Suarez, were strengthened by Cavani's return from suspension.
"(Ecuador) are a good team and they were playing at home where we suffered a lot," Uruguay's coach Oscar Tabarez told reporters.
"The altitude always has an influence, if a player is imprecise he will be more so at altitude. But I'm not going to use that as an excuse, it's not my style."
Chile missed a chance to stay joint top when Colombia came from behind to grab a deserved 1-1 draw in Santiago.
A header from Artur Vidal on the stroke of halt time put the home side ahead but Colombia were the better team in the second period and Real Madrid's James Rodriguez got an equaliser with 67 minutes gone.
Argentina, with only one point from two matches, were supposed to meet Brazil in Buenos Aires but the match was postponed because of torrential rain in the Argentine capital.
The game is now scheduled to take place on Friday at 9pm local time (0000 GMT).
"We met with match officials and the Argentine representatives and there's no way the game can go on," Brazil's assistant coach Gilmar Rinaldi told reporters at the stadium.
"The players could be at risk and the referee decided to postpone the game."
Brazil are sixth in the 10-team group, while Argentina are eighth. Four teams qualify automatically and a fifth goes into a play-off.
Bolivia, who lost their first two matches, lifted themselves off the bottom of the table with a 4-2 win over Venezuela.
Striker Rodrigo Ramallo scored twice, midfielder Juan Arce converted a penalty and Rudy Cardozo hit their fourth in the second half.
Venezuela, who struggled to knit moves together, replied through Mario Rondon and Richard Blanco though had fellow striker Luis Seijas sent off in added time.
Peru face Paraguay on Friday in Lima.
(Reporting by Alexandra Valencia in Quito, Luis Ampuero in Buenos Aires; Writing by Rex Gowar and Andrew Downie; Editing by Greg Stutchbury)
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