Objective To explore the effect of long-term depleted uranium (DU)ingestion on testosterone production in rats, and its involvement mechanism. Methods Male and female rats (F0 and F1 respectively) for 160 days, respectively. The contents of testosterone (T), luteinizing hormone (LH), and follicle stimulating hormone (FSH) in serum were detected in 20 months of F0 generations, and 15 months of F1 generations. RT-PCR was used to analyze the levels of StAR mRNA and P450scc mRNA. Results Compared with the normal control group, the testosterone contents in exposed F0 and F1 generations increased, the lowest was 51.73 U/L, but those of LH and FSH decreased. The expression of StAR mRNA in the low-doze group of F1 generation (StAR/β-actin = 1.35) was up-regulated, down-regulated for other groups.compared with the normal control group (P450scc/β-actin = 0. 313), the expression of P450scc mRNA in the low- and high-dose groups of F0 generation were decreased (P450scc/β-actin = 0.21), and those in the low- and high-dose groups of F1generation were increased (P450scc/β-actin = 0.623) (P ≤ 0.01). Conclusion Long-term DU exposure inhibit the male reproduction by intervening the sexual hormone production through down-regulated the expression of StAR mRNA and P450scc roRNA.