Tributyltins (TBT) are ubiquitous and persistent environmental contaminants that disturb normal endocrine function including gonadal function in humans and marine organisms. TBT was administered through oral route to male Syrian hamsters at daily doses of 50, 100, and 150 ppm/kg for 65 days. Changes in testis morphology, immunohistochemistry of iNOS, 3β-HSD, and 17β-HSD, cholesterol transport receptor, nuclear receptors, and transcription factors were analyzed. TBT treatment affected each of these parameters to significant levels in a dose-dependent manner compared to vehicle treated control. Real-time PCR and protein analyses revealed that expression levels of ApoE and LDL-R mRNA were up-regulated in the testis of TBT-treated animals while the expression levels of SR-B1, LXR, PPARs α, β, and γ, SCAP, SREBP 1 and 2, 3β-HSD, 17β-HSD, CYP17A1, and P450 were down-regulated. Leydig cells were isolated and separated adopting percoll gradient centrifugation under aseptic condition. The viability of Leydig cell was affected by TBT treatment in a dose- and time-dependent manner. Further, the mechanism of action of TBT was ascertained by siRNA transfection of ApoE, which was upregulated, and SREBP, which was down-regulated. These observations led us to infer that exposure to TBT hinders intracellular cholesterol transport resulting in abnormal sex steroid biosynthesis and alteration of steroidogenic enzyme activities. Finally, we could recognize ApoE and SREBP as the major factors regulating genes that control cholesterol biosynthesis and steroidogenesis that ultimately inhibit the synthesis of testosterone. Therefore, ApoE is one of the important molecular targets that can be intercepted in context of male infertility/male contraception.