Tom Bradshaw's double and a Mamadou Thiam penalty-kick condemned Millwall to their first defeat in five Sky Bet Championship games at The Den on Saturday afternoon as Barnsley won 3-1.
Bradshaw opened the scoring in the 40th minute, but Lee Gregory - returning from suspension and making his 150th appearance for The Lions - equalised from the penalty spot in first-half injury time.
However, Bradshaw and Thiam snatched all three points for The Tykes in the second-half.
Millwall boss Neil Harris made two changes from the 2-1 victory over Reading on Tuesday night, with Gregory and Ryan Tunnicliffe - making his first League start - replacing Fred Onyedinma and Shaun Williams in the starting line-up.
The Lions started strongly and could have taken the lead after four minutes, but Gregory's shot was well stopped by Adam Davies.
Barnsley made a game of it as the first-half reached its halfway point, but Brad Potts could only head wide Adam Hammill's inviting cross on 22 minutes.
With 34 minutes on the clock, Jed Wallace returned the ball into the penalty area after it was cleared following George Saville's corner-kick, but the ball trickled just wide after a Barnsley deflection.
An incredible scramble in the box commenced in the 39th minute following a mix-up between Jordan Archer and Byron Webster, but The Tykes could not turn the ball home due to a combination of Millwall defending and weak finishing.
However, seconds later, the away side took the lead. A break away down The Lions' right-hand side saw a cross turned home by Bradshaw to open the scoring.
On the stroke of half-time, Millwall levelled from the penalty spot. Gregory broke away from the Barnsley defence and was cruelly brought down as he shaped to shoot. Matty Pearson should have perhaps seen red for bringing the striker down as the last man, but escaped with just a booking. Gregory, however, stepped up and confidently dispatched his spot-kick to even proceedings.
The first real attempt of the second-half went the way of The Lions, but Wallace's drive was gathered at the second time of asking by Davies.
Barnsley, though, were to break up the other end and re-take the lead through Bradshaw, who converted from close range despite the best efforts of Archer.
With 20 minutes remaining the ball broke to substitute Shane Ferguson at the back post, but the Northern Irishman could only fire over the crossbar. In the 74th minute, Steve Morison met Ferguson's cross, but Davies comfortably claimed the striker's header.
Potts could have sealed the game for Barnsley on 79 minutes, but his free header was brilliantly stopped by Archer.
On 83 minutes, The Tykes added a third from the penalty spot through substitute Thiam.
The Lions huffed and puffed in their attempts to reduce the deficit, but were unable to do so, instead succumbing to their second Den defeat of the campaign.
Millwall: Archer, McLaughlin, Meredith (Onyedinma 81'), Hutchinson, Gregory, Wallace, Webster, Tunnicliffe, Morison, O'Brien (Ferguson 63'), Saville. Subs not used: Martin, Craig, Thompson, Romeo, Cooper.
Scorer: Gregory 45+2' pen
Booking: Wallace, Ferguson
Barnsley: Davies, McCarthy, Williams, Lindsay, Hammill, McGeehan (Bird 80'), Bradshaw (Thiam 72'), Barnes (Hedges 72'), Jackson, Potts, Pearson. Subs not used: Townsend, Ugbo, Macdonald, Fryers.
Scorer: Bradshaw 40', 60', Thiam 83' pen
Booking: Pearson, McGeehan, McCarthy
Referee: Oliver Langford
Attendance: 12,147 (691)