The ubiquitous environmental pollutant Dibutyl Phthalate (DBP) is a known to have anti-androgenic like properties. DBP exposure has been linked to a range of developmental disorders in males such as hypospadias (defect of the penis) and cryptorchidism (undescended testes). Humans are exposed to DBP daily; therefore, it is essential to fully understand the role DBP in the pathogenesis of these developmental disorders. The mechanism of toxicity has yet to be fully established; however, DBP has been shown to reduce testosterone biosynthesis and is thought to disrupt the embryological androgen:estrogen ratio. The present study developed and characterized a LC-540 Leydig cell line model system. This model system was used to investigate the effects of DBP at physiologically relevant concentrations on key genes of testosterone biosynthesis via the use of Nanostring® nCounter technology. The metabolism of DBP was investigated using HPLC to identify if any potentially estrogenic metabolites are produced. The estrogenicity of DBP was investigated using a MCF-7 proliferation assay. The LC-540 model system is able to produce detectable quantities of testosterone with and without external stimulation in culture. The metabolism of DBP did not produce any potentially estrogenic metabolites; however, the LC-540 Phase I and Phase II processes were identified. DBP appears to be slightly estrogenic and promotes growth of MCF-7 cells. Key genes in testosterone biosynthesis are changed following exposure to DBP. The gene that codes for testosterone biosynthesis (hsd17b3) is down-regulated at normal daily exposure concentrations and occupational exposure concentrations. The gene that codes for 17β-estradiol synthesis (cyp19a1) is up-regulated at occupational exposure concentrations. The likely result of these gene changes is the feminization of males which explains the role of DBP in the pathogenesis of hypospadias and cryptorchidism. The present study is the first evidence of dual gene regulatory changes following DBP exposure at physiologically relevant exposure levels. The MCF-7 proliferation assay is the first evidence of DBPs potential estrogenic properties.