The Clarets missed out on a place in the last 16 of the Carabao Cup as Burton pulled off a shock to reach round four for the first time.
The Brewers added their name to Accrington Stanley, Port Vale and Lincoln City in a list of lower division clubs who have made Burnley a cup scalp in recent seasons as Nigel Clough’s side came from behind to send Sean Dyche’s men out at the first hurdle.
European qualification had given the Clarets a belated entry into the competition.
But they failed to take advantage as the League One side recovered from going behind to a Kevin Long goal and survived a handful of missed second-half chances from the Clarets to earn a famous win through record signing Liam Boyce and Jamie Allen’s late strike.
The welcome return of Steven Defour for the midfielder’s first appearance since January was one of six changes made by Burnley boss Dyche as the Clarets looked to utilise their squad strength and build some momentum on the back of a first Premier League win of the season.
But the Clarets never had it all their own way as Burton looked to follow up wins over Shrewsbury and Aston Villa which had brought them through the first two rounds.
Burton boss Clough was twice a League Cup winner as a player with Nottingham Forest and his side gave an early sign of what might be to come with the first chance of the game when Jake Hesketh pulled the ball back for Scott Fraser to shoot straight at Tom Heaton when an effort either side would have taken some keeping out.
That sparked an instant reaction from the Clarets with Dwight McNeil getting behind the Brewers’ defence to pick out Chris Wood for an effort the striker - marginally offside minutes later when converting a perfect Defour through-ball - steered just beyond the far post.
McNeil again impressed and the teenage wideman was involved again as Burnley took the lead five minutes before half-time.
His incisive pass saw Stephen Ward win a corner from a blocked cross and when Johann Berg Gudmundsson delivered, Long rose highest to nod home his third goal for the Clarets.
But with just one goal in it, the tie was never over. Five minutes into the second half the underdogs went close to an equaliser when winger David Templeton cut in and curled an effort across the face of goal.
And after the Clarets failed to double the lead from their best move of the game shortly afterwards – Dimitar Evtimov saving well from Gudmundsson after Defour and Matej Vydra had worked the opening – Burton did get level.
Former Ross County striker Boyce, who once spent a season in Germany with Werder Bremen, had been a handful for most of the evening.
And after Burnley only half stopped a rapid break-away and conceded possession too close to their goal, the Northern Ireland international collected the ball and drilled a deflected shot past Heaton with the aid of a touch off Ashley Westwood from just inside the corner of the box.
In an effort to regain the initiative Dyche sent on Ashley Barnes and the striker had an immediate impact.
With his first touch he collected a pass from Gudmundsson and ran at the Burton defence before crashing a 20-yard shot against a post.
Barnes then nodded down for Westwood to volley over and Wood forced Evtomiv into another smart save as the Clarets stepped up the pace in a bid to avoid the possibility of a penalty shoot-out.
But it was Burton who took that prospect out of the equation when they struck the winner six minutes from time, Boyce playing a part again with a through-ball to Allen who slotted home to write a new chapter in Burton’s history and continue the Clarets’ cup woes.
Burton Albion: Evtimov, Akins, Brayford, Turner, McCrory, Hesketh, Fraser (Sbarra 77), Quinn (Fox 69), Templeton (Sordell 81), Allen, Boyce. Substitutes: Campbell, Miller, Hutchinson, Flannigan.
Burnley: Heaton, Lowton, Long, Mee, Ward, Gudmundsson, Westwood, Defour (Hendricvk 74), McNeil, Vydra (Barnes 71), Wood (Vokes 81). Substitutes: Legzdins, Taylor, Lennon, Bardsley.
Booked: Defour, Long.
Referee: Robert Jones