The incident took place at the Urr Valley Country House in Castle Douglas at the weekend.
Thieves broke into the hotel some time between 22:30 on Saturday and 07:30 on Sunday and made off with the safe and its contents.
Police said the safe was a heavy one and would have needed two people to remove.
They have appealed for anyone who saw anything suspicious in the area to contact them.
Thieves have made off with more than £10,000 when they stole a safe from a south of Scotland hotel.