Fluminense, who went ahead with a 25-minute header by Brazil striker Fred, lost 2-1 away in Porto Alegre after Gremio replied with two goals from playmaker Marquinhos before half-time.
The champions, who have a game in hand, are ninth with 21 points from 15 matches, 13 behind leaders Corinthians and Flamengo, who both drew their 16th games.
Fred had missed their two previous matches having asked to be dropped for "psychological" reasons after being chased by angry fans who caught him drinking late at night in a Rio bar on the eve of a match 10 days ago.
Marquinhos equalised on the half hour when he beat the offside trap to pick up a pass from Lucio and score with a shot that took a deflection on its way into the net.
His second 15 minutes later was a free-kick into the top corner past former Liverpool keeper Diego Cavalieri.
Gremio's first win in five matches lifted the southern side out of the relegation zone to 14th with 18 points.
"We're not on a good run and we're not getting results away from home. It's time we played less nicely to get the results," Fred told reporters, adding that the incident with fans was forgotten.
Corinthians, ahead of Flamengo on goal difference, were held 2-2 at home by Ceara who scored an 85th-minute equaliser through midfielder Rudnei at a corner.
Flamengo, the only unbeaten team, squandered a 2-0 lead having scored two goals through striker Deivid, both laid on by Ronaldinho, in the opening 50 minutes, and drew 2-2 at Figueirense.
Vasco da Gama and Botafogo both won at the weekend, leaving the top five teams separated by just six points.
