Previously, we demonstrated that capsaicin induces tight-junction (TJ) opening in human intestinal Caco-2 cells. In order to clarify the mechanism underlying the TJ opening action of capsaicin, we performed a proteomics study on capsaicin-treated Caco-2 cells. Phosphorylated cofilin was decreased significantly by capsaicin treatment. In addition, capsaicin induced Ca2+ influx in Caco-2 cells and there was a clear correlation between Ca2+) influx and cofilin dephosphorylation (activation). The Ca2+-chelating reagent EGTA blocked the cofilin dephosphorylation induced by both capsaicin and ionomycin, suggesting that the dephosphorylation was mediated by Ca2+ influx. Finally, transepithelial electrical resistance measurements showed that TJ opening accompanied cofilin dephosphorylation. Our data suggest that TJ opening is mediated by cofilin dephosphorylation, which is caused by capsaicin stimuli, including Ca2+ influx. This is the first report of capsaicin action via the dephosphorylation of cofilin in human intestinal cells.