Dibromoacetic acid (DBAA), a by-product formed during disinfection of drinking water, alters spermatogenesis in rats through defective spermiation. The mechanism underlying this toxicity is not fully understood. In this study, gene expression data generated with microarrays from testes were used to generate a mechanistic understanding of DBAA-induced testicular toxicity. Testes were collected from male Sprague-Dawley rats dosed orally for 1 and 4 days with DBAA at 250 mg/kg/day. At both time points, DBAA administration induced delayed spermiation in Stage X tubules and regulated the expression of a small number of genes, including a mild but consistent downregulation of cytochrome P450c17α (CYP17) mRNA, an enzyme expressed by Leydig cells and essential for the production of testicular androgens. Downregulation of CYP17 was confirmed at the protein level and its biological significance was substantiated by demonstrating reduced testicular testosterone levels in DBAA-dosed rats. Furthermore, testosterone production by human chorionic gonadotrophin (hCG)-stimulated rat primary Leydig cells was reduced following treatment with 100 μM DBAA. Collectively, these results indicate that DBAA can directly target rat Leydig cells and downregulate testicular CYP17 expression with a resulting decreased testicular testosterone production. This disruption of testicular steroidogenesis is likely to contribute to the mechanism of failed spermiation observed in rats following exposure to DBAA.