OBJECTIVE To investigate the anti-inflammatory effects of Sirt1 and its roles in steroidogenesis in Leydig cells. DESIGN In vitro cell culture study. SETTING Reproductive medical center. ANIMAL(S) C57BL/6 male mice. INTERVENTION(S) TM3 Leydig cells were treated with proinflammatory cytokines including tumor necrosis factor-α, interleukin-6, or interleukin-1β. MAIN OUTCOMES MEASURE(S) Sirt1 mRNA and protein levels were measured by real-time polymerase chain reaction and Western blotting in TM3 Leydig cells treated with proinflammatory cytokines. The cell viability was determined using MTT and BrdU incorporation assays. The effect of Sirt1 on the coactivation of steroidogenic factor 1 (SF-1) was characterized by coimmunoprecipitation and luciferase reporter assays. RESULT(S) Sirt1 mRNA and protein levels were significantly down-regulated by proinflammatory cytokines treatment in TM3 Leydig cells. Sirt1 agonist or overexpression could efficiently protect cytotoxicity induced by proinflammatory cytokines. Sirt1 promoted steroidogenesis through its coactivation of SF-1. CONCLUSION(S) Sirt1 plays protective roles and promotes steroidogenesis in Leydig cells through its anti-inflammatory actions and coactivation of SF-1.