Objective Previous 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. Methods Adipocytes 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. Results Adipocytes 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. Conclusions A molecular mechanism was proposed in which Ahnak regulates adipocyte differentiation through Smad1 activation.