It came when the Spurs defence only half-cleared an Andy Parkinson corner and from 25 yards out Congolese midfielder J-P Kalala hit a rasping shot through a crowd of players and beyond England keeper Paul Robinson.
The lateness of the goal meant that Spurs had no time to hit back and in truth they created little in the way of chances all night.
Grimsby goalkeeper Steve Mildenhall made a routine save in each half, whereas Robinson had to save well from Parkinson in the first half on a couple of occasions and early in the second period he did just enough to put off Michael Reddy who knocked his shot past the keeper and inches past the post.
Spurs had opened brightly knocking the ball around well and threatening to give the League Two leaders a torrid night, but that threat never materialised and the visitors squandered three good opportunities from free-kicks.
In the second half Spurs pushed Grimsby back and the speed of Aaron Lennon caused the Mariners problems, but there was never a goal threat from Martin Jol's side.
After the final whistle there were marvellous scenes as many of the Grimsby fans poured onto the pitch to mob their heroes. It was as if the team had won promotion rather than take a scalp, albeit a Premiership one.
In contrast a trickle of Spurs fans began to leave the ground after the goal and it certainly wasn't a night to remember for the North London club.
Having put out Derby and Spurs, the Mariners now sail onto round three and will be looking for another big team at home, even though a full house of 8,206 was all they could pack into their tiny Blundell Park ground.