Kaoru Mitoma, who plays for Brighton and Hove Albion, scored in the 23rd minute at Wembley on Tuesday (Wednesday AEDT) to end goalkeeper Jordan Pickford's run of clean sheets for England dating back to October 2024.
It was a first win for fellow World Cup hopefuls Japan over England in four encounters and the second defeat for the world's fourth-ranked side in 12 matches under their German coach.
Captain and prolific scorer Kane was ruled out before the start after picking up an injury in training and England rang the changes from the side that drew 1-1 with Uruguay in a friendly last Friday.
Japan, who also beat Scotland 1-0 on Saturday, have now won their past five games.
England midfielder Cole Palmer was the culprit for losing the ball to Mitoma at the halfway line, with Japan then surging forward on the counter-attack and the winger sidefooting home from Keito Nakamura's cross.
Elliot Anderson came close to an equaliser with a shot that bounced off the bar in the 34th minute, but the visitors could have gone 2-0 up when a deflected effort pinged the woodwork for a corner just before halftime.
There were boos at the break after a lame display by the England players, supposedly fighting for a place on the plane to the World Cup, who failed to step up in their last game on home soil before the finals in the United States, Mexico and Canada.
Phil Foden, playing centrally as a false nine rather than as a winger, made little impact in the first half and was substituted in the second.
Tuchel stuck with his starting line-up until making a quadruple change in the 59th minute, by which time Japan could have been further ahead.
Ritsu Doan squandered one good opportunity, shooting rather than passing to an unmarked teammate, and Junya Ito missed another.
England substitute Marcus Rashford had a chance to equalise in the 78th, but shot straight at keeper Zion Suzuki, and Jarrod Bowen fired wide on the rebound.
Centre-back Marc Guehi, taken off late in the second half and replaced by Harry Maguire, had stepped up as captain after Kane was rested as a precaution following an issue in training.
"Minor injury from basically out of nothing, it just happened," Tuchel told ITV before the game. "He had to step out of training yesterday after 15 minutes."
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