Darlington also repeated the feat of going to a first division club and knocking them out - though it took a penalty shootout for them to overcome Bradford City at Valley Parade.
Northampton beat Norwich, while Notts County knocked out Preston after penalties and Huddersfield ended Derby County's cup run with a 2-1 win.
Wimbledon MK also came unstuck, crashing 2-0 at Wycombe Wanderers.
Another remarkable match was played out at Belle Vue, where Donny Rovers completed an astonishing comeback from 2-0 down to beat nine-man Grimsby Town with the winner coming in the 93rd minute.
Full results:
Barnsley 1 - 2 Blackpool
Bradford 0 - 0 Darlington (Darlo win 5-3 on pens)
Bristol R 0 - 1 Brighton
Cambridge 1 - 2 Gillingham
Cardiff 4 - 1 Leyton Orient
Cheltenham 1 - 2 Queens PR
Chesterfield 0 - 0 Burnley (Burnley win 3-2 on pens)
Colchester 2 - 1 Plymouth
Crewe Alex 2 - 0 Wrexham
Doncaster 3 - 2 Grimsby Town
Huddersfield 2 - 1 Derby County
Lincoln 0 - 1 Stockport
Luton 4 - 1 Yeovil
Macclesfield 1 - 2 Sheff Utd
Millwall 0 - 1 Oxford
Northampton 1 - 0 Norwich
Port Vale 0 - 0 Nottm Forest (Forest win 3-2 on pens)
Preston 0 - 0 Notts County (County win 7-6 on pens)
Rotherham 2 - 1 York City
Scunthorpe 2 - 1 Oldham
Southend 2 - 3 Swindon
Torquay 1 - 1 Crystal Palace (Palace win 3-1 on pens)
Tranmere 1 - 0 Bury
Walsall 2 - 1 Carlisle
Watford 1 - 0 Bournemouth AET
West Brom 4 - 0 Brentford
Wigan 2 - 0 Hull City
Wycombe 2 - 0 Wimbledon MK
