Thyroid hormone signaling has long been implicated in mammalian testicular function, affecting steroidogenesis in testicular Leydig cells. However, its molecular mechanism is not well understood. Here, we investigated the molecular action of thyroid hormone receptor-α (TRα) on mouse testicular steroidogenesis. TRα/thyroid hormone (T3) signaling differentially affected the expression of steroidogenic enzyme genes, mainly regulating their promoter activity. TRα directly regulated the promoter activity of the cytochrome P450 17α-hydroxylase/C17-20 lyase gene, elevating its expression in the presence of T3. TRα also indirectly regulated the expression of steroidogenic enzyme genes, such as steroidogenic acute regulatory protein and 3β-hydroxysteroid dehydrogenase, by modulating the transactivation of Nur77 on steroidogenic enzyme gene promoters through protein-protein interaction. TRα enhanced Nur77 transactivation by excluding histone deacetylases from Nur77 in the absence of T3, whereas liganded TRα inhibited Nur77 transactivation, likely due to interfering with the recruitment of coactivator such as the steroid receptor coactivator-1 to Nur77. Together, these findings suggest a role of TRα/T3 in testicular steroidogenesis and may provide molecular mechanisms for the differential regulation of steroidogenic enzyme genes by thyroid hormone.