Background: Gold nanoparticles (AuNPs) have shown great promise in various biomedical applications, but their effects on male reproductive function remain to be ascertained. The aim of this study was to investigate the uptake, cytotoxicity and testosterone production inhibition of AuNPs in mouse Leydig cells, as well as their accumulation in the testes of male mice and their effects on male reproductive function.Results: AuNPs (5 nm) were able to be internalized into the endosomes/lysosomes of TM3 Leydig cells, induce the formation of autophagosomes, increase the production of reactive oxygen species (ROS), and arrest the cell cycle in S phase, resulting in concentration-dependent cytotoxicity and DNA damage. Interestingly, AuNPs significantly reduced testosterone production in TM3 cells by inhibiting the expression of 17α-hydroxylase, an important enzyme in androgen synthesis. After repeated intravenous injection, AuNPs gradually accumulated and retained in the testes of male BALB/c mice in a dose-dependent manner. One week after withdrawal, the level of plasma testosterone in the 0.5 mg/kg AuNPs group was significantly reduced compared to that in the PBS control group, accompanied by the decreased expression of 17α-hydroxylase in the testes. In addition, AuNPs treatment significantly increased the rate of epididymal sperm malformation, but without affecting fertility.Conclusion: Our results suggest that AuNPs can accumulate in the testes and reduce testosterone production in Leydig cells by down-regulating the expression of 17α-hydroxylase, thus affecting the quality of epididymal sperm.