Abstract: In this research, the effect of 2, 6-diaminopyridinum as a new phenanthroline derivative was studied on the hypophysis-gonad axis, testicular tissue and sperm production in male Balb/C mice. Fifty adult male Balb/C mice were divided in five groups. First group was considered as untreated control. Saline was injected to second group and the remaining three groups received intraperitoneal injection of 15, 20 and 25 mg kg-1 of 2, 6- diaminopyridinum every other day for 20 days. The LD50 was determined to be 35 mg kg-1 body weight. The testicular tissues were studied morphologically and the serum concentration of FSH, LH and testosterone were measured. The results showed that 25 mg kg-1 diaminopyridinum decreased the number of germ cells significantly and serum testosterone level with no change on FSH and LH levels. This study indicates that 25 mg kg-1 of phenanthroline may directly affect testicular tissue causing a lower testosterone level and spermatogenesis in mice.