Multiple anesthesia protocols have been used in the cheetah (Acinonyx jubatus). Twenty healthy, captive cheetahs were immobilized with dexmedetomidine (15.8 ± 1.9 μg/kg), butorphanol (0.22 ± 0.03 mg/kg), and midazolam (0.18 ± 0.03 mg/kg) by intramuscular injection. Induction, recumbency, and recovery times were recorded, and physiologic parameters were monitored. Anesthesia was antagonized with atipamezole (0.125 ± 0.02 mg/kg) and naltrexone (0.1 ± 0.014 mg/kg) intramuscularly. All cheetahs were safely anesthetized with this protocol. Cheetahs were laterally recumbent by 8 ± 3.5 min. Cardiorespiratory values were stable throughout the length of anesthesia. Moderate hypertension, with systolic blood pressure ranging from 178 ± 19.8 mm Hg, was initially observed but decreased over time. There was a statistical decreasing trend in temperature; SpO2; and systolic, mean, and diastolic blood pressure, but not in heart rate and end-tidal CO₂. Recoveries were rapid, with cheetahs standing by 11.3 ± 5.7 min postreversal administration. This is the first report of a dexmedetomidine-butorphanol-midazolam anesthetic combination in cheetahs. Overall, this anesthetic protocol proved to be safe and effective.