Atrazine (ATZ) is a widely used herbicide and is considered an endocrine disruptor of different organisms. However, the molecular interactions of ATZ with biological targets in mammalian endocrine systems are not understood fully. In the present study, we observed that ATZ administration (50, 100 and 200mg/kg) for 3weeks to peripubertal male ICR mice exerted adverse effects on several physiological features; these effects included a significant decrease in the body and liver weights and an increase in the relative testis weight. In addition, the serum testosterone (T) concentration was significantly decreased in all ATZ-treated mice, and the serum estradiol (E2) concentration and aromatase activity were significantly increased in mice exposed to 100 and 200mg/kg ATZ. These results suggest that ATZ exposure affected hormone homeostasis in male mice. We also found that the transcript levels of the steroidogenic enzyme genes p450scc, p450 17α1 and 17β-HSD were significantly reduced in the testes of mice exposed to 100 and 200mg/kg ATZ for 3weeks. Given the results of the present study and previous reports, it is possible that ATZ reduces the T concentration in peripubertal male mice by affecting the transcription of steroidogenic genes, such as p450scc, p450 17a1 and 17β-HSD. This study provides new insights into the mammalian toxicological mechanism of ATZ.