Objective To investigate whether administration of gossypol of 12mg/kg combined with methyltestosterone 20mg/kg and ethinylestradiol 100μg/kg daily for a long term of 24 weeks could affect rat spermatogonial stem cell function. Methods TdTmediated dUTP nick endlabeling (TUNEL) detection, spermatogonial stem cell transplantation and fertility recovery evaluation were carried out. Results The administration of this regimen could induce germ cell going apoptosis; the apoptotic signal positive cells were spermatocyte and spermatid, no spermatogonium was found to be signal positive. Two to three months after transplantation of medicine treated spermatogonial stem cells, elongated rat spermatids were identified in the seminiferous tubules of recipient nude mice. Six weeks after withdrawal of the administration, fertility of the medicine treated rats was recovered completely compared with that of the control group. After a series of tests, litters of the first generation showed to be normal. Conclusion These results indicated that the administration of this regimen would not affect the function of rat spermatogonial