Holders St Mirren were denied a safe passage to the third round at Palmerston Park as Michael Paton's clever finish sealed an unexpected 2-1 victory for Queen of the South.
The Scottish Premiership side were taken to extra time but, despite seeing Paul McGowan miss a penalty, they kept their composure to take the lead with Steven Thompson's header after 95 minutes.
However, their second-tier opponents fought back with Stephen McKenna's fabulous leveller before Paton pinched the win five minutes from time.
Last year's competition saw the Paisley side stun Celtic in the semi-finals before breaking Hearts at Hampden with a 3-2 victory to claim their first major trophy in 26 years.
But Danny Lennon's men headed to Dumfries in worrying form after backing up that March triumph with just one win in the six months that followed.
Their lethargy was evident as Queen's energetic pressing caused them all sorts of problems in the opening period.
While Saints struggled to link up, the hosts were menacing, with Derek Lyle the first to threaten when he fired half a foot over David Cornell's crossbar after just six minutes.
McKenna then sprayed a ball left for Daniel Carmichael to catch but the sprightly winger send his shot wide after cutting past David van Zanten.
A lapse in concentration from St Mirren's Gary Teale let in Paton to fire off a shot which Cornell did well to beat away, before Saints finally conjured an attempt on goal on the stroke of half-time as Gary Harkins strode forward from his own half, shrugging off Paton's attempt at a tackle as he went, before thudding the upright from 20 yards out.
The Buddies started the second half in more confident fashion, with John McGinn watching Calum Antell deny his long-range effort.
With 10 minutes left, Harkins then found the side-netting after Teale's driving run and cross but Queens held out for extra-time.
And there was drama just three minutes into the additional period as Thompson was felled by Paton in the box, only for Antell to deny substitute McGowan's spot-kick.
Saints did not let that blow distract them and as Harkins crossed for Thompson just two minutes later, the former Scotland striker found the net with a downward header.
But Queens responded when McKenna sent a 20-yard screamer past Cornell two minutes before the turn-around to level the match once again.
And Saints' reign as champions was ended five minutes from time when Antell's long punt was misjudged by St Mirren skipper Jim Goodwin, opening the space for Paton to steer a clever lob beyond the stranded Cornell.