Permethrin, the most popular insecticide among the synthetic pyrethroids, has been used worldwide to control a wide range of insects in agriculture, forestry, public health, and homes. Humans may have suffered potential exposure to this compound. The commercial formulation of permethrin contains trans and cis isomers. Here, at the same dosage, we made a comparison of the reproductive effects between these two isomers. Male adult ICR mice were orally administered trans- or cis-permethrin daily for 6 weeks at a dose of 0 or 70 mg/(kg day). In the cis-permethrin exposure group, the caudal epididymal sperm count and sperm motility were significantly reduced, and testosterone levels in testes and plasma also fell. Moreover, cis-permethrin induced abnormal seminiferous tubules in testes and suppressed testicular mRNA expression levels of peripheral benzodiazepine receptor, steroidogenic acute regulatory protein, and the cytochrome P450 side-chain cleavage enzyme. Although such adverse effects were not observed in the trans-permethrin exposure group, testicular and urinary metabolite 3-phenoxybenzoic acid levels in trans-permethrin-exposed mice were about three- and sevenfold higher than those in cis-permethrin-exposed mice, respectively. Furthermore, in vitro, hepatic microsomal hydrolase activity for trans-permethrin was nearly 62-fold higher than that for cis-permethrin. Taken together, the difference in metabolic activity between cis- and trans-permethrin might contribute to the difference in the reproductive toxicity between both isomers.