The purpose of the present study was to investigate the effects of testosterone on proliferative response and IgG production by splenic lymphocytes in vitro. Splenic lymphocytes from rats were cultured in the presence of the indicated doses of testosterone propionate for 72 h. The cell viability was assessed by a trypan blue exclusion test. Proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and supravital stain neutral red assay. Levels of IgG and IL-6 were measured by enzyme-linked immunosorbent assay (ELISA). The proliferative response and IgG production of splenic lymphocytes in the presence of testosterone were lower than those of control cells. Testosterone may inhibit proliferative response and IgG production of splenic lymphocytes. The results also support that androgen may play an important role in immune regulations.