Addicks owner Thomas Sandgaard today opted against offering the 39-year-old a new deal at The Valley, claiming it was a ‘difficult decision’ to make.
His departure came three days after a 4-0 final-day defeat at Ipswich saw them finish in 13th.
The loss signalled the end of an underwhelming season for Charlton, ending the campaign 14 points adrift of the play-offs.
Jackson was appointed caretaker manager in October following Nigel Adkins’ sacking, later earning the position until the end of the season.
Reports claim a new contract would have automatically triggered should his side finish in the top eight – now the club have opted to look elsewhere.
In a club statement, Owner and CEO Sandgaard said: ‘I would like to thank Johnnie for everything he has done for the club.
‘He is a Charlton legend who has given his all for this club as a player, captain, coach, assistant manager and manager.
‘I am grateful for all he has contributed during my time at Charlton, including stabilising us this season when he took over as manager, following our poor start to the campaign.
‘This decision has been a very difficult one to make, Johnnie has done so much for the club, but I felt the time was right to make the change as we continue our preparations for next season.
‘Our search for a new manager has begun. This season wasn’t the season any of us at the club wanted. I have big ambitions for Charlton and it will be important for us to find the right individual that can build on the foundations in place and help us progress on our journey.’
In total, the former Charlton captain took charge of 40 games in all competitions – winning 19.
He becomes the second managerial casualty since the League One season ended on Saturday, after Michael Appleton departed Lincoln, hours following their final match.
Jackson spent eight years as a player with the south London club, amassing 271 appearances and scoring 55 goals, before retiring in 2018.