Nonalcoholic steatohepatitis (NASH) is a chronic liver disease that is manifested clinically by an elevation in hepatic triglycerides, inflammation, and fibrosis. The pathogenesis of NASH remains incompletely understood. Sirtuin 6 (Sirt6), an NAD-dependent deacetylase, has been implicated in the fatty liver disease; however, the underlying molecular mechanisms in the NASH pathogenesis are elusive. The aims of this study were to elucidate the role of hepatic Sirt6 in NASH. Wild-type (WT), liver-specific Sirt6 knockout (KO), hepatic stellate cell (HSC)-specific Sirt6 knockout (HSC-KO), and Sirt6 transgenic (Tg) mice were subjected to a Western diet for 4 weeks. Hepatic phenotypes were characterized and underlying mechanisms were investigated. Remarkably, both the liver-KO and HSC-KO mice developed much worse NASH than the WT mice did whereas the Tg mice were protected from the diet-induced NASH. Our cell signaling analysis revealed that Sirt6 negatively regulates the TGFβ-Smad3 pathway. Biochemical analysis showed a physical interaction between Sirt6 and Smad3 in hepatic stellate cells. Moreover, our molecular data further demonstrated that Sirt6 deacetylates Smad3 at key lysine residues K333 and K378 and attenuates its transcriptional activity induced by TGFβ in the hepatic stellate cells. Our data suggest that SIRT6 plays a critical role in the protection against the NASH development and it may serve as a potential therapeutic target for NASH.