Medical director Dr Geeta Koppikar said that Pansare passed away after his condition worsened around 9pm.
Maharashtra chief minister Devendra Fadnavis has reached Breach Candy hospital.
Govind Pansare was flown to Mumbai from Kolhapur on Friday in air ambulance.
The air ambulance carrying the critically injured Pansare left Kolhapur around 4pm, an official said.
Pansare, who was in the forefront of anti-road toll agitation, and his wife Uma were shot by unidentified persons near their house in Kolhapur on Monday when they were returning from their morning walk.
Police are preparing sketches of the suspects on the basis of information provided by Uma, who is recovering and now able to speak.
Teams of Maharashtra Anti-Terrorism Squad (ATS) and Mumbai police are assisting the Kolhapur police into the probe on the attack on the couple.
(With inputs from PTI)
