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Despite the surprise defeat, Wolfsburg remain on top with a two-point advantage to Hertha Berlin, the runners-up in the Bundesliga standings.
Meanwhile, Energie Cottbus keep the distance with one point to the Bundesliga bottom three.
Both teams entered the first half energetically. In spite of their good motivation, the first half ended without noteworthy shots.
Immediately after beginning of the second half, however, Energie Cottbus showed clearly their ambition to win the match.
In the 72nd minute, Cottbus striker Dimitar Rangelov opened the score with a header from the right side of the goal-area to the centre of the goal.
Wolfsburg tried to fight back in the game, but it was again Cottbus who took the initiative. In the 87th minute, Cottbus midfielder Ervin Skela doubled the lead with a shot from the right side of the box.
The defeat, the biggest surprise in the Bundesliga's 29th round, has ended the Wolves' straight win of 10 matches.
"We have wasted a big chance, in this way one can not win," Wolfsburg coach Felix Magath said after the game.
Also on Sunday, the game between two bottom teams Borussia Moenchengladbach and Arminia Bielefeld ended with a 1-1 draw.
Both teams thus remain in the Bundesliga drop zone.