The historic artifact was stolen at some point between 12pm on March 17 and 10am on March 18 from Aberdour Castle in the village of Easter Aberdour, Fife.
Police are now appealing for information from the public in finding the 16th century mill stone which would have been used for grinding wheat or other grains.
The object would require at least two people to lift it, according to the police.
A spokesperson for Historic Environment Scotland (HES), said: “We can confirm that a millstone has been stolen from Aberdour Castle.
“This 16th century millstone is one of the few remnants of water-powered mill that once stood in the castle grounds next to the Dour Burn, and an important piece of the story of the site.
"Grain was ground into flour between this stone and a larger flat one underneath. A wooden spindle, powered by a waterwheel, turned the top stone.
“The loss of such an artefact is a heritage crime which robs us of a priceless piece of our past, and we would urge anyone with information regarding this incident to contact Police Scotland on 101.”
A Police Scotland spokesperson said: “Sadly, despite our checks, the issues at Aberdour Castle continue.
"This 500-year-old mill stone was stolen from there between 12pm on 17/03 and 10am on 18/03.
"It would take at least two people to lift it.
The news of the theft comes after a series of anti-social behaviour in the area.
Police commented that ‘damage’ occurred at the 13th century castle two weeks ago.
Chief Inspector Yvonne Stenhouse said: “We are aware of ongoing issues with youths engaging in anti-social behaviour which is concerning, particularly in the midst of a pandemic when people are being told to stay at home.
"Local officers are working with partners to address these issues and proactively stop this activity.
“I would urge anyone with information or concerns regarding anti-social behaviour to contact Police Scotland through 101 or speak to officers out on patrol. Alternatively, you can call Crimestoppers anonymously on 0800 555 111.”