Capsaicin is the major pungent component in hot chili pepper. It is known that capsaicin induces the increase of energy metabolism through adrenaline activation. The aim of this study was to investigate the mechanism underlying the effect of capsaicin on energy metabolism in human intestinal epithelial cells (Caco-2). To clarify the mechanism, we performed proteomics, real time-PCR, ATP measurement, and MTT in this study. Glycolytic enzymes, namely, phosphoglycerate mutase (PGM) and triosephosphate isomerase (TPI), were overexpressed in capsaicin-treated Caco-2 cells. mRNA expression levels of TPI and PGM were also increased in capsaicin-treated Caco-2 cells. Furthermore, intracellular adenosine triphosphate (ATP) content, which is the end product of glycolysis, was increased. To demonstrate the intracellular ATP production and ATP accumulation, the viability of Caco-2 cells incubated with glucose free medium was measured. The viability of capsaicin pretreated cells was higher than that of untreated cells. All these observations strongly indicate that capsaicin increases energy metabolism in human epithelial cells through the activation of glycolytic enzymes.