ObjectivePrevious reports have indicated that Ahnak‐deficient mice were protected from high‐fat diet‐induced obesity. However, the molecular mechanism in which Ahnak mediates adipocyte differentiation and high‐fat diet‐induced obesity is unclear.MethodsAdipocytes from Ahnak knockout (Ahnak−/−) mice and knockdown of Ahnak in C3H10T1/2 were used to investigate the function of Ahnak in adipocyte differentiation. Ahnak‐induced adipocyte differentiation was analyzed by Oil Red O staining.ResultsAdipocytes from Ahnak−/− mice were smaller than those from wild‐type mice. Silencing of Ahnak in C3H10T1/2 and adipose tissue‐derived mesenchymal stem cells (ADSCs) from Ahnak−/− mice showed severely impaired adipocyte differentiation. Down‐regulation of Ahnak in C3H10T1/2 cells and ADSCs from Ahnak−/− mice attenuated the phosphorylation and nuclear localization of Smad1 in response to BMP2, whereas Ahnak overexpression in 3T3‐L1 cells significantly increased Smad1 activation. Because PPARγ is a well‐known transcriptional factor in adipocyte differentiation, the PPARγ expression in Ahnak‐mediated adipocyte differentiation was investigated. Transfection of C3H10T1/2 cells with Ahnak siRNA resulted in reduced PPARγ expression apparently through inhibited binding of Smad1 to the Smad1‐binding site in the PPARγ promoter. These results suggest that Ahnak regulates adipogenesis by regulating Smad1‐dependent PPARγ expression.ConclusionsA molecular mechanism was proposed in which Ahnak regulates adipocyte differentiation through Smad1 activation.