King left the field in the third quarter of the Saints' 13.15 (93) to 8.14 (62) win over North Melbourne - his first AFL appearance in 757 days - due to hamstring tightness.
Scans confirmed a strain to King's left hamstring, with St Kilda drawing a line through his season on Monday.
"It's an incredibly disappointing outcome for Max, who has done everything possible throughout his rehabilitation to put himself in the best position to return," general manager of football Lenny Hayes said.
"He's an incredibly important part of our football club. We'll continue to wrap our arms around him and provide all the support he needs as he works through this latest setback.
"Our focus now turns to ensuring Max undertakes a full pre-season to allow him to get back and play consistent senior football in 2027."
Coach Ross Lyon had defended the decision to play King in his first AFL game since June 30, 2024, having battled knee injuries as well as soft-tissue concerns.
The 26-year-old played two VFL games earlier in the year and suffered a hamstring injury in one of those, pushing his comeback past the two-year mark.
The 10th-placed Saints remain firmly in the finals wildcard mix, but losing King, who is on a monster contract until the end of the 2032 season, is a blow to their September hopes.
Earlier on Monday, captain Callum Wilkie said he wasn't surprised to see King come straight back into the AFL team.
"It's not like we rushed him back, he did the hammy 12 weeks ago, and it wasn't a 12-week hammy, it was about a four-five week hammy," Wilkie told SEN.
"They took a very conservative approach with it, and built his training loads and conditioning up over the last 12 weeks, but especially the last month or so where he was hitting game loads at training.
"I was playing on him and saw what he can do, he was ready to come back and it was great to have him back out there playing with us."
King left the ground early and wasn't involved in St Kilda's team song after the win.
"I messaged him saying we love him and we're right behind him and whatever this is doesn't change who he is and what he means to our football club," Wilkie said.
There was also bad news for GWS dynamo Finn Callaghan, who will require season-ending ankle surgery.
The exciting midfielder sprained his ankle late in the 51-point derby loss to Sydney.
"Following scans and a specialist consultation, Callaghan and the club's medical team have opted for surgery to repair the damage," the Giants said.
"Callaghan is expected to be sidelined for approximately 12 weeks."
GWS sit 12th, a win and significant percentage outside the wildcard places.
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