Tottenham’s hopes of reaching the Champions league last 16 were dealt a blow after Kevin Kampl’s 65th minute strike handed Bayer Leverkusen a 1-0 victory at Wembley.
On a night when Mauricio Pochettino asked his players and the club's fans to embrace the Wembley experience, Spurs, who lost Mousa Dembele to injury in the first-half, looked sluggish and they were eventually made to pay by Kampl's winner.
The goal stunned Wembley into silence and though Spurs went close to an equaliser in the final stages with Eric Dier's free-kick rattling the crossbar, Leverkusen held on to spark joyous scenes among the travelling support.
More Wembley woe means Tottenham drop to third in Group E behind Monaco - who beat CSKA Moscow 3-0 in the group's other game - and their Bundesliga victors, with two games left of the group stage.
It is now six games without a win in all competitions for Pochettino's men, who face a north London derby against Arsenal in the Premier League on Sunday.
Pochettino made two changes to the side that drew 1-1 all with Leicester at the weekend with Danny Rose and Vincent Janssen making way for Ben Davies and Moussa Sissoko.
Spurs, hoping to make amends under the arch after an opening defeat to Monaco, struggled to settle in the early stages with Bayer Leverkusen always looking a threat on the counter.
In particular, Javier Hernandez looked lively on his return to England and the former Manchester United striker got in behind the Spurs defence in the 16th minute, but his cross was intercepted by Kyle Walker at the far post.
The hosts were forced into an early substitution on the half hour mark with Janssen replacing Dembele and the change nearly brought a goal as Christian Eriksen stung the palms of Bernd Leno with a powerful shot from 20 yards.
The best chance of the half though fell to the visitors in the 43rd minute when Walker was caught in possession by Julian Brandt.
The 20-year-old drove into the box only to be stopped by Jan Vertonghen's last-ditch tackle. However, the ball rebounded to Hernandez, who attempted to pick out the bottom corner from 14 yards but the ball deflected of Vertonghen's foot and wide.
There was still time for Spurs to put the ball in the net after Dele Alli swept home the rebound after Janssen's shot was saved, but the effort was ruled out after the Dutchman was adjudged to have fouled Jonathan Tah in the build-up.
Spurs started the second half with renewed vigour and they thought they should have had a penalty when Alli was clipped in the box by Omer Toprak, but the appeals were turned down as Swedish referee Jonas Eriksson allowed play to go on with Eriksen firing wide.
Eriksen was then denied by Leno before Janssen shot wide but Leverkusen always looked dangerous on the break.
Vertonghen and Hugo Lloris were needed to stop Hernandez breaking the deadlock in the 61st minute but Leverkusen were ahead four minutes later.
Tottenham failed to close down Charles Aranguiz on the edge of the penalty area and his shot took two deflections on the way through to Kampl, who finished from close range to put the visitors ahead.
The hosts' only real chance of an equaliser came nine minutes from time as Dier struck a free-kick against the crossbar. The ball rebounded into the six-yard area but Leverkusen scrambled clear and held on for a famous victory.