Perfluorooctanesulfonate (PFOS) was produced and used by various industries and in consumer products. Because of its persistence, it is ubiquitous in air, water, soil, wildlife, and humans. Although the adverse effects of PFOS on male fertility have been reported, the underlying mechanisms have not yet been elucidated. Here, for the first time, the effects of PFOS on testicular signaling, such as gonadotropin, growth hormone, insulin-like growth factor, and inhibins/activins were shown to be directly related to male subfertility. Sexually mature 8-wk-old CD1 male mice were administered by gavages in corn oil daily with 0, 1, 5, or 10 mg/kg PFOS for 7, 14, or 21 days. Serum concentrations of testosterone and epididymal sperm counts were significantly lower in the mice after 21 days of the exposure to the highest dose compared with the controls. The expression levels of testicular receptors for gonadotropin, growth hormone, and insulin-like growth factor 1 were considerably reduced on Day 21 in mice exposed daily to 10 or 5 mg/kg PFOS. The transcript levels of the subunits of the testicular factors (i.e., inhibins and activins), Inha, Inhba, and Inhbb, were significantly lower on Day 21 of daily exposure to 10, 5, or 1 mg/kg PFOS. The mRNA expression levels of steroidogenic enzymes (i.e., StAR, CYP11A1, CYP17A1, 3beta-HSD, and 17beta-HSD) were notably reduced. Therefore, PFOS-elicited subfertility in male mice is manifested as progressive deterioration of testicular signaling.