Organotin compounds, such as tributyltin (TBT), that have been used as antifouling biocides can induce masculinization in female mollusks. However, few studies addressing the effects of TBT on fishes have been reported. The present study was conducted to investigate the effects of TBT at environmentally relevant concentrations (1,10, and 100 ng/L) on testicular development in Sebastiscus marmoratus and to gain insight into its mechanism of action. After exposure for 48 d, the gonadosomatic index had decreased in a dose-dependent manner. Although the testosterone levels in the testes were elevated and the 17β-estradiol levels were decreased, spermatogenesis was suppressed. Moreover, γ-glutamyl transpeptidase activity (which is used as a Sertoli cell marker) was decreased in a dose-dependent manner after TBT exposure, and serious interstitial fibrosis was observed in the interlobular septa of the testes in the 100 ng/L TBT test group. Increases in the retinoid × receptors and peroxisome proliferator activated receptor γ expression and the progressive enlargement of lipid droplets in the testes were observed after TBT exposure. Estrogen receptor α levels in the testes of the fish exposed to TBT decreased in a dose-dependent manner. The reduction of estrogen receptor α mRNA resulted from the decrease of 17β-estradiol levels, and the progressive enlargement of lipid droplets may have contributed to the dysfunction of the Sertoli cells, which then disrupted spermatogenesis.