The heavy defeat was the Tykes' 10th in 17 games this season and left them bottom of the Championship table, six points adrift of safety.
Flitcroft, 39, was previously assistant manager to Keith Hill at Oakwell but took sole charge on December 29 last year after impressing in a caretaker role following Hill's departure.
He steered Barnsley to Championship safety last season on the final day following a 2-2 draw at Huddersfield Town, but his side have struggled once again this term.
Technical coach Martin Scott and strength and conditioning coach Guy Proctor have also parted company with the club.
Assistant manager Micky Mellon has been placed in caretaker charge of first-team affairs, assisted by development coach Paul Heckingbottom.
A club statement read: "Barnsley Football Club can confirm first-team manager David Flitcroft has been relieved of his position at the football club.
"The club would like to place on record their appreciation and thanks for what David, Martin and Guy have achieved."
Former Portsmouth, Blackpool and Blackburn manager Michael Appleton was installed as Sky Bet's early favourite to replace Flitcroft.