Jason Puncheon struck 10 minutes from time to send League Two Barnet into the FA Cup fourth round at the expense of Championship high-flyers Colchester.
The midfielder drilled in a stunning winner from the edge of the area to send the minnows through and set up a clash against Plymouth or Peterborough.
It had started so well for Colchester when Jamie Cureton snatched the opener.
But Ismail Yakubu bundled home the equaliser to set up a tense finish and Puncheon sealed it with a fine winner.
The victory was all the more remarkable considering the Bees, languishing in 14th spot in League Two, lay 56 places below sixth-placed Championship outfit Colchester.
And the results owed much to the stoic performance of the home defence, which resisted a string of U's attacks before Cureton fired in from the right-hand side of the box in the 35th minute.
Richard Garcia, Greg Halford and Kemal Izzet all wasted decent chances from close range to extend the visitors' lead and they paid the price when, having resisted another surge at the start of the second half, Barnet levelled.
Although Dean Sinclair and Adam Birchall had been denied by fine saves by Us keeper Dean Gerken, it was against the run of play that they levelled, the ball ricocheting in off Yakubu after good work from Liam Hatch.
And, after Kevin McLeod had hit the post for the visitors, Puncheon wrapped up a fine win with a goal deserving of winning an entertaining Cup tie.
Barnet: Harrison, Bailey, King, Hendon, Sinclair, Graham, Nicolau, Yakubu, Puncheon, Cogan (Hatch 61), Birchall.
Subs Not Used: Flitney, Grazioli, Hessenthaler, Warhurst.
Booked: Hendon, Hatch.
Goals: Yakubu 62, Puncheon 80.
Colchester: Gerken, Halford, Brown, Watson, Cureton, Izzet (Jackson 59), Iwelumo (Guy 58), Baldwin, Garcia, Barker, McLeod (Duguid 78).
Subs Not Used: Davison, White.
Goals: Cureton 35.
Att: 3,075.
Ref: T Kettle (Berkshire).