Objective To investigate the effect of testosterone on mitotic orientation in rat prostate epithelial cells and the relative differential gene expression. Methods Twenty SPF male SD rats were divided into 2 groups at random and then subjected to castration. One group of rats was administrated with testosterone 3.7 mg daily for 30 days and the control group was only injected with olive oil. Microscopic analysis was performed using immunohistochemistry. Differential gene expression analysis was conducted by gene microarray and RT-PCR techniques. Results In the testosterone-administrated group, there was a significant mitosis orientation parallel to the basement membrane. But in the control group, mitosis orientation was oriented perpendicular to the basement membrane. Using the gene microarray and RT-PCR techniques, the cell proliferation genes such as Ran, Tgm4 and Wnt2 in Wnt signal pathway were up-regulated in the testosterone group. Conversely, suppressor cell proliferation genes such as Dkk3 and Fas were down-regulated. Conclusions Mitotic orientation of prostate epithelia cells is changed after testosterone administration. Wnt signal pathway and AR singling pathway also have an influence on the mitosis orientation and cell proliferation.