Objective To establish a method of mouse artificial ejaculation for repeating collection of sperm from mice, which is carried out in many experiments with semen. Methods Three ways were compared including massage with hand (MH), artificial vagina pump (AVP) and electro-ejaculation (EJ). Results AVP and EJ could induce ejaculation, and their success rate, average semen quantity and motility showed no significant difference. Repeated tests got the same data. Mouse ejaculation was divided into four stages with different cell quantity. MH produced no ejaculation. Conclusion AVP and EJ were feasible ways for mouse ejaculation.