Di Canio blamed the 1-0 Johnstone's Paint Trophy loss on defender Aden Flint, declaring that the 23-year-old's mind was elsewhere.
"Flint came in tonight as if he was on holiday and this is not acceptable," he told reporters.
"He has to take the responsibility. We lost because of him."
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The rant comes just days after the Italian laid into goalkeeper Wes Foderingham, who he substituted just 21 minutes into a 4-1 loss to Preston North End.
"He's the worst professional I've ever seen," he said after the defeat.
"It wasn't only the mistake, which can happen to everyone. It was the arrogance later on when he started moaning to the others. That was the worst thing for me.
"If he doesn't come out and say sorry to the fans for his professionalism, then he is out of my team."
Di Canio has a reputation for public disputes with his players, having endured spats with defender Paul Caddis and striker Leon Clarke, who eventually left the club after the manager vowed never to play him again.
The Wiltshire side, who gained automatic promotion by winning League Two last year, currently sit eighth in the English third tier with eight points.