Permethrin, a popular synthetic pyrethroid insecticide used to control noxious insects in agriculture, forestry, households, horticulture and public health throughout the world, poses risks of environmental exposure. Here we evaluate the reproductive toxicity of cis-permethrin in adult male ICR mice that were orally administered cis-permethrin (0, 35 or 70 mg/kg/day) for 6 wk. Caudal epididymal sperm count and sperm motility in the treated groups were statistically reduced in a dose-dependent manner. Testicular testosterone production and plasma testosterone concentration were decreased with an increase in luteinizing hormone (LH) significantly and dose-dependently and a significant regression was observed between testosterone levels and cis-permethrin residues in individual mice testes after exposure. However, no significant changes were observed in body, reproductive organ absolute and relative weights, sperm morphology, and plasma follicle stimulating hormone (FSH) concentration after cis-permethrin treatment. Moreover, cis-permethrin exposure significantly diminished the testicular mitochondrial mRNA expression levels of peripheral benzodiazepine receptor (PBR), steroidogenic acute regulatory protein (StAR), and cytochrome P450 side-chain cleavage (P450scc) enzyme and protein expression levels of StAR and P450scc. At the electron microscopic level, mitochondrial membrane damage was found in Leydig cells of the exposed mouse testis. Our results suggest that insecticide permethrin may cause the mitochondrial membrane impairment in Leydig cells and disrupt testosterone biosynthesis by diminishing the delivery of cholesterol into the mitochondria and decreasing the conversion of cholesterol to pregnenolone in the cells, thus reducing subsequent testosterone production.