The Hatters were sunk by another late goal as an injury-time strike from Stuart Sinclair gave Bristol Rovers all three points.
In a game in which the Town struggled to create many clear-cut chances in front of goal, the Pirates pinched all three points with a goal at the death to leave Kenilworth Road stunned.
Sinclair raced through in behind the Town defence to slam a shot past Mark Tyler to leave the Hatters to reflect on a first defeat of the season.
The Hatters, who were unchanged from Saturday’s 2-2 draw with Oxford, began on the front-foot with Paddy McCourt pulling the strings in midfield. The Northern Ireland international fired the first shot of the evening on seven minutes but his effort from the edge of the box flew wide of the post.
Rovers, promoted from the Conference via the Play-Offs last season, arrived at Kenilworth Road protecting an incredible unbeaten record away from home in league competition stretching back to September last year.
And slowly the visitors worked their way into the game. On 16 minutes left-back Lee Brown’s curling left-foot free-kick was blocked by the Town wall after Daniel Leadbitter had been pulled down on the edge of the box by Luke Wilkinson.
And a minute later it was hearts in mouths time for the Town when Jermaine Easter’s volley from the edge of the penalty area took a huge deflection and dropped inches wide of the target with Mark Tyler beaten.
As the Hatters huffed and puffed in search of a way through the Rovers rearguard, it was the visitors that continued to look the more likely side to open the scoring. Sinclair arched an effort goalwards that was cleared by Nathan Doyle on 38 minutes, and moments later Tom Lockyer planted a header just wide following a deep left-wing cross.
Rovers then came even closer to taking the lead on 40 minutes, and the Hatters had Tyler to thank for an excellent stop to keep out Brown’s first-time effort after the visiting left-back had ghosted in at the back post to meet a low right-wing cross.
The visitors continued in the same vein after the break as the Town struggled to test Aaron Chapman in the visitors’ goal.
The Pirates were denied again by Tyler six minutes after the restart when the Town cleared a chance off the line following a melee in the penalty box.
With the Hatters failing to create any chance of note in a frustrating period of play, John Still introduced Jonathan Smith and Pelly Ruddock Mpanzu in a bid to up the ante as the final 20 minutes approached.
There was a brief rally from the Town with 15 minutes to go with McCourt almost conjuring up a chance for Mackail-Smith on 74 minutes only for Chapman to cut out the winger’s low cross.
Still then sent on Jack Marriott for Mackail-Smith, but as the game entered four minutes of injury time, it looked as the though the match would end in stalemate.
However, Rovers had other ideas and in the final minute of four additional shown, they won it. Sinclair latched onto a ball over the Town of the Town defence and he took one touch before firing past Tyler to send the visiting support into ecstasy.
There was no time left for the Hatters to find an equaliser – and that was that.
Town: Tyler; O’Donnell, Griffiths, McNulty, Wilkinson, Cuthbert, Doyle (sub Smith 64), McGeehan (sub Ruddock 69), McCourt, Mackail-Smith (sub Marriott 74), Benson. Subs not used: Potts, Lawless, McQuoid, Justham.
Attendance: 8,061, incLuding 553 from Bristol Rovers