Consumption of spicy foods has been reported to convey thermogenic properties. Thus, ingredients in these foods could be considered as potential agents for prevention of a positive energy balance and obesity. Capsaicin in particular is one of the main capsaicinoids, which is a pungent principle of red pepper, and is also utilized as a medicine. In this study, 2-dimensional gel electrophoresis (2-DE) was carried out to identify differential expression of liver proteins in rats fed with a high-fat diet (HFD) in response to capsaicin treatments. In addition, immunoblot analysis of some liver proteins was performed for validation of proteomic analysis and suggestions of a molecular action of capsaicin. Results of animal experiments revealed that weight gain of rats in the HFD + Cap group was decreased by 8% compared to the HFD control group. In our search for potential proteins associated with thermo-genesis and lipid metabolism, we analyzed differential expression patterns in rat liver using 2-DE. Proteomic analysis of liver samples demonstrated that approximately 120 spots were differentially expressed from a total of 950 matched spots, of which 23 spots have been identified by peptide mass finger printing using MALDI-TOF mass spectrometry. Protein levels of UCP2 and FAS were decreased, whereas those of p-AMPK, p-ACC, and CPT-1 were increased by capsaicin administration. These data suggest that the effect of capsaicin on energy expenditure and fatty acid oxidation in rat liver might be mediated through activation of the AMPK-ACC-malonyl-CoA metabolic signaling pathway.