The 37-year-old will hang up his boots after captaining the Spireites in their final League Two fixture of the season against Exeter at the Proact Stadium.
It was thought Saturday's game would just be his last for the Blues after being told he would not be offered a new contract in the summer.
But on Friday afternoon Lester stated his intention to call time on his playing career altogether.
He said: "It has been a tough decision but I've got some opportunities coming up to observe coaching around the world. I'll get an insight into some international sports psychologists, using their expertise. I'm also doing some coaching in America.
"I've got a year before my son starts school, which is why I've decided to retire and make the most of the opportunities that are there."
Chesterfield manager Paul Cook paid tribute to Lester, saying: "There's no doubt Jack will go down as a legend at this club. He's been a fantastic servant to this club.
"I'd love him to be able to sign off with a goal in front of the Kop and then see everyone give him the send off that he deserves. I wish Jack every success for the future."
Lester, who joined Chesterfield on a free transfer in the summer of 2007 following the end of his second spell at Nottingham Forest, has scored 92 goals in 218 appearances for the Spireites.
He began his career at Grimsby, where he spent time on loan at Doncaster, and also played for Sheffield United in between his stints at the City Ground.