Hoffenheim poached an early lead when Bosnia striker Sejad Salihovic curled in a free-kick after just nine minutes to shock Dortmund and the champions' attack had a rare off-day as they failed to impose themselves on a determined defence.
Dortmund's rising teenage Germany star Mario Götze, who orchestrated his country's 3-2 friendly win over Brazil in midweek, had a quiet game and was taken off by coach Jürgen Klopp after 54 minutes.
Having beaten Hamburg 3-1 last weekend, Dortmund showed none of the self-assurance which secured that victory, despite Klopp re-shuffling his attacking midfield with three second-half changes.
"Hoffenheim got the victory because they showed greater passion," said Klopp. "In spite of that, we still could have still managed to win the game. Over 40 to 50 minutes, we played well and created a number of chances to score.
"The defeat feels really bad as I didn't have the feeling we couldn't have picked up anything here in terms of points."
Having been shocked at home to Borussia Mönchengladbach last Sunday, Bayern Munich picked up their first win as Brazil midfielder Luiz Gustavo scored in the 91st minute against 2009 champions VfL Wolfsburg.
French play-maker Franck Ribery provided the final pass and Gustavo drilled his shot into the top left-hand corner.
"We were playing against a team who had their heads held high," said Bayern coach Jupp Heynckes, referring to Wolfsburg's 3-0 win at Cologne last weekend.
"We were very happy to get a goal, even if it came in added time. After the opening defeat to Mönchengladbach, it was exceptionally important to get a point here."
Bayern were at near full strength, although Dutch winger Arjen Robben was missing because of a back and adductor problem.
Wolfsburg had the ball in the Munich net in the first half, thanks to a header from ex-Germany striker Patrick Helmes, but the effort was ruled offside, leading coach Felix Magath to call for video referees.
"If four referees are not able to assess the situation properly, then it's time for television referees," fumed Magath after replays showed Helmes was not offside.
Schalke 04 bounced back from last Saturday's defeat at VfB Stuttgart with an impressive 5-1 win at home to Cologne as Dutch striker Klaas-Jan Huntelaar scored a hat-trick.
Hanover and Mainz both picked up their second wins of the season. Hanover - who host Sevilla in a Europa League play-off on Thursday - picked up a 2-1 win at Nuremberg, while Mainz enjoyed a 2-1 win at Freiburg to go top of the table on goal difference.
Hertha Berlin picked up a point in a 2-2 draw at Hamburg.
VfB Stuttgart and ten-man Borussia Mönchengladbach shared a point on Saturday evening.
'Gladbach had defender Roel Brouwers sent off for a second yellow card while Belgium defender Filip Daems netted a second-half penalty for the hosts before Stuttgart's Cacau equalized.
Champions League qualifiers Bayer Leverkusen are chasing their first win of the season at home to Werder Bremen on Sunday, while newly-promoted Augsburg are at Kaiserslautern.