Introduction & Objective: Diethylstilbestrol (DES) is one estrogenal compound that used for treatment of breast cancer and prostate cancer. The aim of this study is to investigate the effects of DES in spermatogenesis in male mature Balb/C mice. Materials and Methods: In this research, 32 mature mice has been used about weighting 35 - 40 g and age of 80 – 90 days which the mice are divided in four group with eight- numbers which includes one control group, one sham group and two experimental group . The experimental group and sham group has received 10 mg/kg and 20 mg/kg DES for 21 days as daily.Results: The experimental II group has received 0.02 mg/kg DES for 21 days as daily. At last, parameters such as body weight, weight of left and right testis, the number of spermatogonia, spermatocyte, spermatid, sperm, Leydig and sertoli cells as well as rate of LH and testosterone hormone concentration. It has been studies results observed, reduction in number of spermatogonia, spermatocyte, spermatid and sperm cells.Conclusion: But, hasn’t significient decrease body weight, weight of left and right testis, the number of Leydig and sertoli. Also hasn’t significient decrease concentration of LH hormone. But, it was demonstrated decrease in concentration level of testosterone hormone as comparision with control group. It can concluded, that use of DES may decreases rate of testosterone hormone secretion and cells number of spermatogonia, spermatocyte and spermatid that probably, affected fertility in Balb/C mice.