The exotic pet, which is 12 months old, has a vulnerable shell and the shop appealed for information on Facebook, saying it was concerned about its "specific care requirements".
The animal was taken from Lathom Pets and Aquatics in Ormskirk on Tuesday afternoon, Lancashire Police said.
The shop's owner said CCTV showed a man taking the tortoise - which needs calcium supplements - out of the tank.
Four people had entered the pet shop together, co-owner Catherine Broxholme said.
"I was serving, chatting to the group and getting change out of the till for a woman I was serving. The tortoise tank is just a few feet away from the till. We only have five tortoises and when I checked later there were only four."
CCTV footage confirmed a man had taken the tortoise, she said.
Ms Broxholme said she was concerned for the exotic pet's welfare, adding: "They have specific care needs. We are a welfare-oriented pet shop, making sure the pets go to good homes.
"The tortoise is about 12 months old and its shell is still vulnerable and it needs calcium supplements. We just want to get the tortoise back safe."
Police said the tortoise was valued at Â£120.
A baby tortoise has been stolen from a pet shop in Lancashire.