Grimsby's Jean-Paul Kamudimba Kalala struck with only a minute left to send Spurs crashing out of the Carling Cup at an ecstatic Blundell Park.
Kamudimba Kalala fired home a powerful right-foot strike from 20 yards to give Russell Slade's League Two leaders a deserved victory.
It was a major embarrassment for a below-par Spurs side boasting five England players and 10 internationals.
Robbie Keane almost equalised in injury time, but Steve Mildenhall saved well.
Despite the strength of the Spurs side, Grimsby refused to be over-awed by their more illustrious opponents and striker Michael Reddy's all-action style soon proved a real menace to the Londoners.
Spurs should have taken the lead after 22 minutes when Andy Reid raced clear inside the box, but Mildenhall was alert and made a crucial block. Two minutes later Grimsby came even closer to finding the net.
Kalala, an international with the Democratic Republic of Congo, delivered a cross from the right and after Gary Jones nodded back across the face of goal, Reddy was inches away from making crucial contact.
England goalkeeper Paul Robinson then saved brilliantly from Andy Parkinson as Grimsby threatened again, turning away a long-range curling drive. Grimsby continued to press after the restart and Reddy had a golden opportunity to make the all-important breakthrough.
He found himself clear of Tottenham's defence with only Robinson to beat but the forward could only watchin agony as his attempted lob sailed harmlessly wide.
But with time running out, Kalala struck from distance after Parkinson's corner was headed out to him on the edge of the box to put Mariners fans in dreamland.
Grimsby: Mildenhall, McDermott, Whittle, Robert Jones, Croft, Cohen (Barwick 83), Kamudimba Kalala, Bolland, Parkinson, Reddy, Gary Jones (Gritton 45).
Subs Not Used: Ramsden, Newey, Toner.
Goals: Kamudimba Kalala 89.
Tottenham: Robinson, Stalteri, Naybet, King, Lee, Jenas, Carrick (Davis 67), Brown, Reid (Lennon 61), Defoe, Keane.
Subs Not Used: Cerny, Kelly, Bunjevcevic.
Ref: G Laws (Tyne & Wear).