Obesity is associated with a number of pathological disorders such as non-insulin-dependent diabetes, hypertension, hyperlipidemia, and cardiovascular diseases. alpha-Lipoic acid (LA) has been demonstrated to activate the insulin signaling pathway and to exert insulin-like actions in adipose and muscle cells. Based on this similarity LA is expected to promote adipogenesis in pre-adipocytes. Here, however, we report that LA inhibited differentiation of 3T3-L1 pre-adipocytes induced by a hormonal mixture or troglitazone. Northern blot analysis of cells demonstrated that this inhibition was accompanied with attenuated expression of adipocyte-specific fatty acid-binding protein and lipoprotein lipase. Electrophoretic mobility shift assay and Western blot analysis of cells demonstrated that LA modulates transcriptional activity and/or expression of a set of anti- or pro-adipogenic transcription factors. LA treatment of 3T3-L1 pre-adipocytes also resulted in prolonged activation of major mitogen-activated protein kinase signaling pathways but showed little or no effect on the activity of the insulin receptor/Akt signaling pathway. These findings suggest that LA inhibits insulin or the hormonal mixture-induced differentiation of 3T3-L1 pre-adipocytes by modulating activity and/or expression of pro- or anti-adipogenic transcription factors mainly through activating the MAPK pathways.