The potentially life-saving piece of equipment is believed to have been taken on Wednesday afternoon, from its base at Collydean Baptist Church.
The defibrillator, which cost around £1300, was one of two installed in north Glenrothes by the community council.
“It’s totally senseless that anyone would do it,” said community council member Denise Wallace.
“The good thing is that it can’t be used unless it’s needing to be used.”
This is the second time the defibrillator has been stolen.
Ms Wallace added: “People are checking gardens, woodland, and bins for it.
“We found it the last time.”
Sergeant Kirk Donnelly of Glenrothes Community Policing Team, said: “Public access defibrillators are there to help save lives. They are a vital tool which any member of the public can use to assist in a cardiac arrest prior the arrival of medically trained staff. This theft is not only thoughtless but has now removed a vital life saving tool from the community.
“I am keen to hear from anyone who might have been in the area of Torphins Avenue that may have seen anyone acting suspiciously, or alternatively if anyone knows who is responsible for this to contact police.”
Contact Police Scotland on 101 quoting reference 3960 of 11th July or Crimestoppers anonymously on 0800 555 111.