The defibrillator was taken from a secure cabinet outside the Co-op on High Street, Burntisland, at about 02:30 on Monday.
It was installed by Burntisland First Aid Services Trust in May 2015 after £850 was raised by the local community.
No-one has had to use the kit, which administers an electric shock to the heart during or after a cardiac arrest.
The defibrillator was previously stolen three months after being installed but was soon returned.
PC Pat Devaney, of Police Scotland, said: "This piece of equipment is vital as it is used to provide medical assistance to people suffering heart attacks and other heart-related emergencies.
"I would urge whoever took it to return it to its rightful place."
Thieves have stolen a potentially life-saving piece of equipment from outside a shop in Fife.