Nonylphenol is the primary final biodegradation product of nonylphenol polyethoxylate (NPE), a non-ionic surfactant that is frequently incorporated into pesticide and detergent formulation. Recent researchers have hypothesized that environmental/ occupational exposure to nonylphenol poses adverse effects on reproductive system of humans and wildlife species. During our study, in vivo and in vitro experiments were performed to examine the effect of nonylphenol on testosterone biosynthesis of rat Leydig cells. In experiment in vivo, serum testosterone (T) as well as luteinizing hormone (LH) levels were detected after animals had been treated with different doses (0 mg/kg/day, 125 mg/kg/day, and 250 mg/kg/day) of nonylphenol for 50 days by gavage, and the final result revealed that testosterone level dramatically declined at the dose of 250 mg/kg/day, while LH level ascended at the dose of 125 mg/kg/day and 250 mg/kg/day. In experiment in vitro, primary cultured Leydig cells were exposed to nonylphenol for 48 h, including low concentrations (0 mg/L, 0.0011 mg/L, 0.0033 mg/L, 0.0055 mg/L, 0.011 mg/L, 0.022 mg/L) and higher concentrations (0.11 mg/L, 0.55 mg/L, 1.1 mg/L, 1.65 mg/L, 2.2 mg/L, 2.75 mg/L, 3.3 mg/L, 5.5 mg/L). Increase of testosterone levels was observed at low concentrations of nonylphenol while reduction was detected at higher concentrations.