Zookeepers say they last saw 13-year-old Butti on Monday afternoon in The Loft, a hands-on exhibit where visitors can handle Butti and his brother Tutti.
Officials say someone might have walked off with the Indian Star tortoise - he's the size of a grapefruit and weighs about a pound.
The Colorado Springs Gazette reported Tuesday that zookeepers are most concerned about Butti's health because he requires a special diet of certain vegetables.
Indian Star tortoises typically live 10 to 15 years.
Butti has a history of upper respiratory infections. Without his caretakers, he might develop gastrointestinal problems, osteoporosis or kidney failure.