To evaluate sedative and physiological effects of low dose intramuscular (IM) alfaxalone, six healthy rabbits were administered single IM doses of alfaxalone at 1mg/kg (IM1), 2.5 mg/kg (IM2.5), or 5 mg/kg (IM5) with a minimum of 7-day washout period. Sedative effects were subjectively evaluated using a composite measure scoring system (maximum sedation score of 16) and pulse rate, respiratory rate, non-invasive blood pressure, and percutaneous oxygen-hemoglobin saturation were measured before and after IM alfaxalone. Loss of righting reflex (LRR) was achieved in all rabbits after IM2.5 and IM5 treatments but in only three rabbits after IM1 treatment. Median (interquartile range) times to LRR were 16 min (15–17), 6 min (6–6), and 4 min (4–4), and median durations of LRR were 0.5 min (0–7), 22.5 min (19–27), and 53 min (48–58) after IM1, IM2.5, and IM5 treatments, respectively. The duration of LRR after IM5 treatment was significantly longer than those after IM1and IM2.5 treatments ( P <0.01). Median value of total sedation scores peaked at 10 min [score 3.5 (3–4)], from 10 min [score 13.5 (12–14)] to 15 min [score 13.5 (12–14)], and from 10 min [score 15 (12–15)] to 15 min [score 15 (14–15)] after IM1, IM2.5, and IM5 treatments, respectively. No rabbit showed circulatory depression and apnea although respiratory rate decreased after IM 2.5 and IM5 treatments. In conclusion, alfaxalone produced a dose-dependent sedative effect and a deep sedation was achieved by alfaxalone at 2.5 mg/kg IM in rabbits.