Alcohol-induced intestinal barrier dysfunction is a major contributor to alcoholic liver disease (ALD). Forkhead box protein O1 (FoxO1) is a member of the mammalian forkhead box O class (FoxO) subfamily that regulates a wide array of cellular processes. In the present study, we used both an alcohol-fed mouse model and an alcohol-treated Caco-2 intestinal epithelial cell monolayer in vitro model to investigate whether FoxO1 is involved in alcohol-induced intestinal barrier dysfunction. We found that chronic alcohol exposure to mice significantly increased both mRNA and protein levels of FoxO1 in all the examined intestinal segments with the most remarkable changes in the ileum. Alcohol treatment increased mRNA and protein levels of FoxO1 and promoted nuclear translocation of FoxO1 in Caco-2 cells. Furthermore, alcohol treatment with Caco-2 cells resulted in a significant decrease in the epithelial transepithelial electrical resistance (TEER) value, which was attenuated by knockdown of FoxO1 expression. In conclusion, our data suggest that activation of FoxO1 is likely to be a novel mechanism contributing to the deleterious effects of alcohol on intestinal barrier function.