The Addicks stalwart had been in permanent charge in south-east London since December after overseeing seven victories from 10 League One matches in his role as caretaker boss following the dismissal of Nigel Adkins two months earlier.
Jackson guided Charlton to the safety of mid-table and a 13th-placed finish in the third tier, having been languishing in the relegation zone following a run of just two wins in 13 games when he first took the temporary reins.
However, that was evidently not enough to earn him a new deal, with the former long-serving midfielder and first-team coach now exiting after a difficult season finished on a real downbeat note with a 4-0 drubbing by Ipswich at Portman Road on Saturday.
The decision to part company with Jackson is likely to prove highly divisive among the Charlton fanbase, with the 39-year-old remaining a hugely popular figure among supporters having been at The Valley since 2010 – winning two promotions during that time.
Former Tottenham man Jackson made 279 appearances for the club before hanging up his boots in 2018 and moving into coaching.
“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,” said Charlton owner Thomas Sandgaard.
“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.”