Mauricio Pochettino's side went into the Europa League Round of 32 second leg a goal down to Belgium's eighth-placed side and needed to produce a winning result at Wembley.
But it all went wrong as Gent forced a 2-2 draw against 10-man Spurs to advance to the last 16 for the first time ever with a 3-2 aggregate.
Spurs started brightly and sparked the massive Wembley crowd inside 10 minutes when Christian Eriksen raced onto a ball over the top from Eric Dier, before calmly slotting beyond Lovre Kalinic from an angle.
The home side were looking to attack at will but soon enough they found themselves behind the eight ball when Gent scored down the other end.
Late in the first half, there was a moment of madness from midfielder Dele Alli who produced a wild challenge on Brecht Dejaegere which prompted referee Jorge de Sousa to brandish a straight red.
That made life even more difficult for Spurs who required two goals in the second half with just 10 men to advance to the next stage.
Spurs were probing and probing as the minutes ticked on and they managed to get themselves right back in the contest on 61 minutes when Victor Wanyama produced a quality curling finish.
This sprung the Wembley crowd into life and inspired the Spurs players to create clear-cut chances but they were unable to make the most of their dominance.
Gent hit Tottenham on the counter late in proceedings which saw the ball kindly for substitute Jeremy Perbet to poke home for his second goal in two outings against the North Londoners, wrapping up proceedings.
It was another failure for Tottenham at English football's headquarters but fortunately they still have the FA Cup and second place in the Premier League to play for.
2016/17 UEFA Europa League - Round of 32 second leg
Tottenham Hotspur (ENG): 2 (Christian Eriksen 10', Victor Wanyama 61', red card: Dele Alli 39')
KAA Gent (BEL): 2 (Harry Kane (OG) 20', Jeremy Perbet 82')
*Gent win 3-2 on aggregate
Wembley Stadium, London, England - 90,000