Few studies evaluate anesthesia in black-tailed prairie dogs (Cynomys ludovicianus). Isoflurane inhalant anesthesia is used in this species most commonly, but injectable protocols are poorly described. Here we compared the physiologic effects, including anesthetic depth, vital signs, and hematologic changes, of anesthetic protocols using isoflurane or a combination of dexmedetomidine, ketamine, and midazolam in black-tailed prairie dogs. In a randomized, complete crossover study design, intact male black-tailed prairie dogs (n = 9; age, 6 mo) were anesthetized by using a combination of dexmedetomidine (0.25 mg/kg IM), ketamine (40 mg/kg IM), and midazolam (1.5 mg/kg IM). For reversal, atipamezole (0.15 mg/kg) and flumazenil (0.05 mg/kg) were administered 45 min after induction. For comparison, isoflurane was administered at 5% in 100% oxygen at 5 L/min in an anesthetic induction chamber, followed by maintenance isoflurane 2% in 2 L/min oxygen through a tight-fitting facemask for 45 min. Induction and recovery time, respiratory rate, heart rate, body temperature, SpO₂, indirect blood pressure, and reflexes were monitored every 5 min during the anesthetic period. Blood samples for venous blood gases, PCV, and refractometric total protein were obtained from the cranial vena cava at 5 min and 45 min. Both protocols appeared to achieve safe and effective anesthesia. Except for blood pressure, all vital signs differed between the 2 treatments. Isoflurane anesthesia resulted in a slightly longer induction and lower respiratory rate and body temperature but increased likelihood of absent reflexes. DKM anesthesia resulted in a faster induction and less hypothermia but also prolonged recovery and lower heart rate and SpO₂ readings. These findings suggest that isoflurane provides a more stable and consistent anesthetic plane, whereas dexmedetomidine-ketamine-midazolam anesthesia may be an effective alternative for short procedures that require fast induction and limited analgesia.