We aimed at elucidating the roles of transforming growth factor (TGF)-beta and Smad3 signaling in adipocyte differentiation (adipogenesis) and in the pathogenesis of obesity. TGF-beta/Smad3 signaling in white adipose tissue (WAT) was determined in genetically obese (ob/ob) mice. The effect of TGF-beta on adipogenesis was evaluated in mouse embryonic fibroblasts (MEF) isolated both from WT controls and Smad3 KO mice by Oil red-O staining and gene expression analysis. Phenotypic analyses of high-fat diet (HFD)-induced obesity in Smad3 KO mice compared to WT controls were performed. TGF-beta/Smad3 signaling was elevated in WAT from ob/ob mice compared to the controls. TGF-beta significantly inhibited adipogenesis in MEF, but the inhibitory effects of TGF-beta on adipogenesis were partially abolished in MEF from Smad3 KO mice. TGF-beta inhibited adipogenesis independent from the Wnt and beta-catenin pathway. Smad3 KO mice were protected against HFD-induced insulin resistance. The size of adipocytes from Smad3 KO mice on the HFD was significantly smaller compared to the controls. In conclusion, the TGF-beta/Smad3 signaling pathway plays key roles not only in adipogenesis but also in development of insulin resistance. (C) 2011 Elsevier Inc. All rights reserved.