Impairment in gut epithelial integrity and barrier function is associated with many diseases. The homeostasis of intestinal barrier is based on a delicate regulation of epithelial proliferation and differentiation. AMP-activated protein kinase (AMPK) is a master regulator of energy metabolism, and cellular metabolites are intrinsically involved in epigenetic modifications governing cell differentiation. We aimed to evaluate the regulatory role of AMPK on intestinal epithelial development and barrier function. In this study, AMPK activator (AICAR) improved the barrier function of Caco-2 cells as indicated by increased transepithelial electrical resistance and reduced paracellular FITC-dextran permeability; consistently, AICAR enhanced epithelial differentiation and tight junction formation. Transfection of Caco-2 cells with AMPK WT plasmid, which enhances AMPK activity, improved epithelial barrier function and epithelial differentiation, while K45R (AMPK dominant negative mutant) impaired; these changes were correlated with the expression of caudal type homeobox 2 (CDX2), the key transcription factor committing cells to intestinal epithelial lineage. CDX2 deficiency abolished intestinal differentiation promoted by AMPK activation. Mechanistically, AMPK inactivation was associated with polycomb repressive complex 2 regulated enrichment of H3K27me3, the inhibitory histone modification, and lysine-specific histone demethylase-1-mediated reduction of H3K4me3, a permissive histone modification. Those histone modifications provide a mechanistic link between AMPK and CDX2 expression. Consistently, epithelial AMPK knockout in vivo reduced CDX2 expression, impaired intestinal barrier function, integrity and ultrastructure of tight junction, and epithelial cell migration, promoted intestinal proliferation and exaggerated dextran sulfate sodium-induced colitis. In summary, AMPK enhances intestinal barrier function and epithelial differentiation via promoting CDX2 expression, which is partially mediated by altered histone modifications in the Cdx2 promoter.