Academy boss Andy Awford has taken temporary charge of the first team for Saturday's away game against Newport County at Rodney Parade.
A club statement said"We would like to thank Richie for all his work and wish him well for the future,".
Barker met with Portsmouth chairman Iain McInnes and chief executive Mark Catlin on Wednesday night at Fratton Park and was told of his fate on Thursday.
The Pompey Supporters' Trust, which owns around 51% of the club, added that the prospect of relegation from the Football League was "unacceptable".
"In the position we are in, we believe this is the right decision for the club," said PST chairman Ashley Brown.
"Ultimately football is a results business and we haven't seen the progress we'd hoped for under Richie. We also considered the view of our fans and decided, as a group, that this is the right decision."
Former Crawley manager Barker, who lost his job at the League One Club, took over from Guy Whittingham at Fratton Park on 9 December and lost eight of his 20 games.
Awford, who was also caretaker for three games when Whittingham was sacked, has seven games to prevent the club from a third successive relegation.
Awford has told BBC Radio Solent "We all need to be together, the staff, the players, the board and the fans,"