James Beattie's side came from behind to beat the Championship outfit on a night of surprises, but last season's beaten finalists were knocked out by Huddersfield
The League Two club came from behind to overcome their Championship counterparts in the first round of the competition on Tuesday, adding to the woes of Middlesbrough manager Tony Mowbray following their 2-1 league defeat to Leicester City on Saturday.
Lukas Jutkiewicz netted the opener for Middlesbrough nine minutes in, before Marcus Carver levelled five minutes before the interval.
Accrington sealed a famous victory at the Riverside 10 minutes from time, midfielder Piero Mingoia rifling home from a tight angle.
The result makes it just five competitive wins in 2013 for Mowbray's men, but Beattie's charges can celebrate on the back of a spirited display.
"We had a little bit of luck, which you need, but the lads worked so hard and thoroughly deserved their victory," the Accrington player-manager told the BBC.
"We put in a magnificent performance. I'm proud of the players and I'm sure everyone else at the club is too.
"They defended with their lives and deserve the plaudits they are going to get. The hope is always for a financial windfall in the next round. The chairman and the lads want a big draw and they deserve to get one."
Accrington were not the only surprise winners on Tuesday.
Danny Crow scored an extra-time double as League Two newcomers Newport County won 3-1 at second-tier Brighton & Hove Albion.
A late goal from Ryan Williams gave Morecambe a shock triumph over Wolves, while Rotherham United edged past local rivals Sheffield Wednesday 2-1.
Sheffield United also succumbed to defeat, falling 2-1 at home to Burton Albion, while League One Stevenage comfortably overcame Championship outfit Ipswich Town 2-0.
Elsewhere there were comfortable wins for Bolton, Nottingham Forest and Burnley, who beat Shrewsbury, Hartlepool United and York City respectively.
However, last year's finalists Bradford City were knocked out 2-1 by neighbours Huddersfield Town.