Medetomidine/vatinoxan (Zenalpha®) is a novel anesthetic combination used as a sedative and analgesic in dogs. Vatinoxan minimizes adverse cardiopulmonary effects associated with medetomidine administration while preserving sedation and analgesia. In this study, we evaluated the clinical safety and efficacy of 3 dosage combinations of Zenalpha with ketamine and buprenorphine extended release (ER) as compared with xylazine with ketamine and buprenorphine-ER for anesthesia of C57BL/6J mice. We hypothesized that anesthesia with 0.5 mg/kg of Zenalpha would more reliably provide a surgical anesthetic plane, lower mortality, and fewer adverse physiologic effects as compared with anesthesia with 8 mg/kg of xylazine. Ten-week-old male and female C57BL/6J mice were randomly administered 1 of 4 anesthetic cocktails subcutaneously: ketamine (80 mg/kg) and buprenorphine-ER (0.5 mg/kg) with 1) xylazine (8 mg/kg; XKB); 2) Zenalpha (0.25 mg/kg; ZKB/0.25); 3) Zenalpha (0.5 mg/kg; ZKB/0.5); or 4) Zenalpha (1.0 mg/kg; ZKB/1.0). Following drug administration, we assessed the anesthesia induction time by measuring the time to loss of righting reflex and loss of paw withdrawal reflex (PWR). Upon reaching a loss of righting reflex, physiologic parameters including heart rate, respiratory rate, oxygen saturation, indirect mean arterial blood pressure, body temperature, jaw tone, and skin color were monitored every 5 min. Thirty minutes after anesthetic drug administration (TA), atipamezole (1 mg/kg SC) was administered. Recovery time was determined through time until return of PWR, righting reflex, and ambulation. Mice were monitored for 3 d postanesthesia. Results included: 1) ZKB anesthesia caused loss of PWR in a dose-dependent manner; 2) physiologic parameters were similar between XKB and ZKB mice by TA in 100% O₂; 3) ZKB groups took longer to recover and had a 20% to 30% mortality rate in the mid-to-high dosage groups. We conclude that anesthesia with 0.5 mg/kg of Zenalpha more reliably produced a surgical anesthetic plane but also led to decreased mean arterial pressure and increased mortality as compared with anesthesia with 8 mg/kg of xylazine. We recommend using Zenalpha (0.25 to 1.0 mg/kg) with 80 mg/kg ketamine and 0.5 mg/kg buprenorphine-ER to provide general anesthesia in C57BL/6 mice, along with supplemental 100% oxygen and atipamezole.