Despite their insulin-sensitizing effects, antidiabetic thiazolidinedione (TZD) drugs possess strong adipogenic properties as activators of peroxisomal proliferator activator receptor γ. Increased adipogenesis is a risk factor for type 2 diabetes. This study aims to investigate these paradoxical effects at the molecular level. We used proteomics technologies to identify differentially expressed proteins and GenMAPP/MAPP-finder software to obtain biological pathways influenced by pioglitazone and rosiglitazone in 3T3-L1 cells. 155 differentially expressed proteins were observed, of which 102 were identified. Pioglitazone- and rosiglitazone-treated 3T3-L1 cells showed increased rates of triglyceride accumulation and induction of proteins involved in intracellular fatty acid transport, glycerol 3-phosphate synthesis and gluconeogenesis from non-carbohydrate substrates. Simultaneously, TZDs influenced tricarboxylic acid cycle proteins and induced the complete fatty acid beta-oxidation pathway together with oxidative phosphorylation proteins. In conclusion, TZDs change the proteome of 3T3-L1 cells to enable increased TG storage but also fatty acid beta-oxidation. Together, these results indicate that TZDs transform pre-adipocytes into cells with insulin-sensitizing properties that may contribute to the therapeutic effectiveness of these drugs. Copyright © 2006 by New Century Health Publishers, LLC. All rights of reproduction in any form reserved.