The effect of nanoparticle-rich diesel exhaust (NR-DE) on the testicular function and factors related with the biosynthesis of testosterone gene expression were investigated in mice. Male C57BL/Jcl mice were exposed to clean air, low-dose NR-DE (Low NR-DE), high-dose NR-DE (High NR-DE) or filtered diesel exhaust (F-DE) for 8 weeks. We found that the mice exposed to High NR-DE had significantly higher testosterone levels than those in the control and F-DE groups. To determine the effects of NR-DE on testicular testosterone production, interstitial cells dissected from the male mice which were exposed to NR-DE, F-DE, or clean air for 8 weeks were incubated with or without human chorionic gonadotropin (hCG; 0.1 IU/mL) for 4 h. The concentrations of testosterone in the culture media were measured. The testosterone production was significantly increased in with or without hCG of High NR-DE exposed group, and significantly decreased in both with or without hCG of F-DE exposed groups. Moreover, several genes, which is associated with testicular cholesterol synthesis, HMG-CoA, LDL-R, SR-B1, PBR, and P450scc, P450 17α, and 17β-HSD were determined in the testis of adult male mice. The results showed High NR-DE exposure significantly increased the expression of these genes. Whereas, the levels in the F-DE exposure group returned to those in the control group, implicating that the nanoparticles in DE contribute to the observed reproductive toxicity. We conclude that enhancement of testosterone biosynthesis by NR-DE exposure may be regulated by increasing testicular enzymes of testosterone biosynthesis.