To compare four methods for developing immune-suppressed mouse models induced by cyclophos-phamide (CP) with different doses and ways,and choose the appropriate animal model of immunosuppression to evaluate the immunotoxicity of a substance. Methods The female BALB/c mice were assigned to five groups:the control group with distilled water daily gavage; CP gavage group with 40mg/kg BW/d by gavage; CP ip-1group with 40mg/kg BW/d by intraperitoneally injection (ip); CP ip-2group with 80mg/kg BW/d in the first three days by ip and once a week in the following period; CP ip-3group with 200mg/kg BW 24h before the end point by ip. The study lasted for 30days. ResultsThe immune organ weights,the number of leucocyte and the percentage of lymphocytes in peripheral blood,IgA and IgG levels in serum, the percentage of B cells in peripheral blood, the plaque forming cells in spleen, 24h footpad thickness change, LPS- and ConA-induced splenocyte proliferation in four cyclophosphamide groups were significantly lower than those in control group (P＜0.05). The percentages of neutrophils in peripheral blood in four cyclophosphamide groups were significantly higher than those in control group (P＜0.05). However, the percentage of NK cells in peripheral blood in CP gavage group and CP ip-1group were significantly higher than that in control group, and the percentage of Th cells and the ratio of CD4⁺ to CD8⁺ in CP ip-2group and CP ip-3group were higher than those in control group (P＜0.05). In addition, the body weight and liver relative weight in CP gavage group and CP ip-1group were lower than those in control group, the levels of alanine aminotransferase, glucose and triglyceride changed significantly (P＜0.05). There was no significant difference between CP ip-2group and CP ip-3group except the body weight and haematology parameters such as mean corpuscular hemoglobin concentration and red blood cell distribution width (P＞0.05). Conclusion The four ways of CP administration could induce immunosuppression in BALB/c mice, however the appropriate animal model of immunosuppression may be established by intraperitoneal injection of 200mg/kg BW CP 24h before the end point.