Increasing dose of Mithotrexate (25, 50, 100, 150, 200 mg/kg) were tested on Swiss strain mouse by one intramuscular injection, and their effects were studied (15) days later. The mean values of total spermatogonia and those of them undergoing mitotic division (in the metaphase) were evaluated in seminiferous tubular cross section, and the mitotic index (MI) of the spermatogonia was also calculated. Statistical analysis was performed by using IBM-Spss 19 program and for mean values comparison (one way ANOVA: post Hos Test, Multiple Comparison, and Tukey HSD) was used, while mitotic index was calculated by using Target Variable. Our results showed that Methotrexate injection didn’t lead to a significant decrease in spermatogonia mean values in the tested animals compared to the control, while it led to a dose dependent gradual decrease in the mean values of the spermatogonia undergoing mitotic division , and the greatest reduction was noticed in the two high doses: 150, 200, mg/kg (7.10±9.4, 3.65±2.15 respectively) compared to the control (37.40±2.26), and which led also to a severe reduction in the mitotic index (20.53%, 6.36% in the doses: 100, 200 mg/kg respectively) compared to the control (61.79%).