The collision happened just after midnight in the area of Wellington Road 109 and Side Road 16, west of Arthur.
Police said a transport truck and an SUV collided head-on.
The driver of the SUV was airlifted to hospital with life-threatening injuries and police announced on Wednesday that he has since died.
He has been identified as Glen Nix of Orangeville.
The driver of the truck was not injured.
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OPP are still investigating the cause of the collision and are asking any witnesses to call 1-888-310-1122.
Arthur is about 40 kilometres northwest of Guelph.
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