Fish are commonly anesthetized with MS-222 (tricaine methanesulfonate), a sodium-channel-blocker used as an immersion anesthetic, but its mechanism of action as a general anesthetic is uncertain. Alfaxalone is a neurosteroid that acts at the GABA_A receptors. Alfaxalone has been evaluated and was deemed successful as an immersion agent in koi carp. Alfaxalone is an effective intramuscular anesthetic in multiple species. A reliable intramuscular anesthetic in fish would be useful in multiple settings. The purpose of this study was to investigate alfaxalone as an intramuscular injectable anesthetic agent in koi carp (Cyprinus carpio). Eight koi carp were utilized in a crossover design. In each trial, six fish received 1 mg/kg, 5 mg/kg, or 10 mg/kg of alfaxalone intramuscularly. They were assessed every 15 min for opercular rate and sedation score. The sedation score was based on a visual scale from 0 to 5, 0 indicating no response and 5 indicating absent righting reflex and anesthesia. Anesthetized koi were placed on a fish anesthesia delivery system (FADS). Time to anesthesia/recovery was recorded and heart rate was recorded every 15 min. Anesthesia was achieved in 0/6, 1/6, and 5/6 fish at 1, 5, and 10 mg/kg, respectively. Duration of anesthesia for one fish at 5 mg/kg was 2 hr. At 10 mg/kg, median anesthesia duration was 6.5 (3–10) hr. At 10 mg/kg, prolonged apnea (2–3 hr) was observed in 3/6 fish, 2/3 died under anesthesia, and 1/3 recovered 10 hr post-injection. Median peak sedation scores were 1.5, 2.5, and 5, at 1, 5, and 10 mg/kg, respectively. A dosage of 10 mg/kg alfaxalone resulted in 33% mortality. The duration of anesthesia and opercular rate were unpredictable. Due to variation in response despite consistent conditions, as well as risk of mortality, intramuscular alfaxalone cannot be recommended for anesthesia in koi carp.