Christian Eriksen and Lucas Moura missed in the shootout as Tottenham were dumped out of the EFL Cup by League Two outfit Colchester United, who won 4-3 on penalties after holding their Premier League opponents to a 0-0 draw.
Mauricio Pochettino's men failed to break down their fourth-tier opponents despite having Dele Alli, Lucas and Victor Wanyama in their starting line-up and were punished by John McGreal's side, who knocked Crystal Palace out on penalties in the previous round.
Promising young players Oliver Skipp and Troy Parrott were given rare starts but found themselves embroiled in a real battle with a Colchester side who defended resolutely and gave their Premier League visitors more than the odd scare at JobServe Community Stadium.
Eriksen, on as a second-half substitute, had his penalty saved before Lucas hit the crossbar to prompt wild celebrations among the home fans, but Tottenham are now without a victory in five away games across all competitions.
Teenage forward Parrott dragged a low effort wide after 15 minutes before Lucas blazed a powerful shot over the crossbar.
Lucas then bent a free-kick towards goal from the edge of the box, drawing a good diving save from United goalkeeper Dean Gerken, but Tottenham went into half-time frustrated after Colchester largely contained their Premier League visitors.
Kyle Walker-Peters hit the post with a cross-cum-shot as the pressure on Colchester grew, but the hosts threatened on the break – Paris Cowan-Hall sending a raking shot narrowly wide from 20 yards.
Courtney Senior, Colchester's liveliest player, gave Spurs another scare when his drive from outside the box bounced just wide as the game went to penalties.
Eriksen stepped up first and Gerken made a brilliant one-handed save down to his right, but Jevani Brown's attempted panenka backfired allowing Paulo Gazzaniga an easy save.
But Lucas crashed Tottenham's fifth penalty onto the crossbar and Colchester's supporters stormed onto the field to celebrate a historic night for their club.
What does it mean? Homesick Spurs slip up again
Tottenham's last away victory in a competitive fixture came at Ajax in the UEFA Champions League back in May and Pochettino needs to find a way to start winning on the road before his season is derailed.
Sanchez saves Spurs from an even worse result
Colchester made numerous forays into Tottenham's half, but the assured presence of Davinson Sanchez at the heart of the visitors' defence ensured nothing came of them in 90 minutes.
Alli forces Pochettino's hand
Any plans Pochettino had to give Son Heung-min and Eriksen a rest went out of the window as they were subbed on after 66 minutes, in no small part due to Alli's failure to get the job of picking Colchester apart done without them.
Pochettino's men return to the sanctuary of Tottenham Hotspur Stadium for a clash with Southampton in the Premier League on Sunday (AEST), while Colchester come back down to earth as they travel to Macclesfield Town in League Two.