Nonylphenol (NP) is an important environmental toxicant and potential endocrine disrupting chemical. It is also the primary final biodegradation product of nonylphenol polyethoxylate, a non-ionic surfactant that is frequently incorporated into detergent formulation. Several studies have reported adverse effects of NP on the male reproductive system, including hormonal changes and testicular abnormalities. However, its mechanisms are not clearly understood. In the present study, experiments were performed to examine the effects of 4-nonylphenol on the testosterone production in mice Leydig cells. Interstitial (Leydig) cells were cultured with addition of 0.04, 0.2, 1.0, 2.5 and 5 μg/mL of 4-nonylphenol and compared with control. Concentrations of testosterone in the media were determined using enzyme linked immunosorbent assay (ELISA). Hormone production significantly (P<0.05) increased at 1.0, 2.5 and 5.0 μg/mL of 4-nonylphenol concentrations. Results of the study indicated dose-dependent increases in testosterone production of interstitial (Leydig) cells following a 48 h in vitro 4-nonylphenol exposure. Further investigations are required to establish the biological significance and possible reproductive implications.