Mule, the hybrid of horse and donkey, is sterile.Quite rarely, there are some female mules are fertile.However, there is not any fertile male mule have been reported till now.In this study, we analyzed biopsies of testis from four male mules and compared them with the normal male horse and donkey.Fibroblasts were isolated from the testis and chromosome specimens were prepared by the fibroblasts.Besides, HE staining and immunoflorescent histochemistry were used to analyze and compare the difference between the testes.These studies provide some cellular and tissue evidence for the sterility of mule and pave the way for researching the mechanisms of mules reproductive regulation.