Fenitrothion is a broad-spectrum organophosphate insecticide. Recently, it has been reported to exert androgenic or anti-androgenic activity in in vitro and in vivo screening assays, although the effects appear equivocal in vivo. To provide a conclusive and comprehensive evaluation of fenitrothion, especially regarding its anti-androgenic activity in the reproductive and endocrine systems, we conducted a one-generation reproductive toxicity study at appropriately toxic dose levels with a number of sensitive endpoints for endocrine disruption. Fenitrothion was administered to Crj:CD(SD)IGS parental animals (P) at concentrations of 10, 20, and 60 ppm in the diet for 10 weeks prior to mating, and throughout mating, gestation and lactation. Their offspring (F1) were exposed from weaning until maturation at the age of 10 weeks. In the P generation, brain cholinesterase activity was remarkably reduced in the 60 ppm males and in the 20 and 60 ppm females. Reproductive performance, organ weights, histopathology, and sperm analytical parameters were not affected. In the F1 generation, no general toxicity or effects on anogenital distance, retention of areolae/nipples, onset of puberty, organ weights, histopathological findings, and sperm parameters were observed. In conclusion, fenitrothion had no effects on the reproductive or endocrine systems of the P and F1 generations, even at toxic doses that markedly suppressed brain cholinesterase activity in P animals. The results suggest that fenitrothion at in-use levels in the environment is unlikely to cause disruption of human endocrine systems.