AMP activated protein kinase (AMPK), a sensor of cellular energy status in all eukaryotic cells, is activated by LKB1-dependent phosphorylation. Recent studies indicate that activated LKB1 induces polarity in epithelial cells and that this polarization is accompanied by the formation of tight junction structures. We wished to determine whether AMPK also contributes to the assembly of tight junctions in the epithelial cell polarization process. We found that AMPK is activated during calcium-induced tight junction assembly. Activation of AMPK by 5-aminoimidazole-4-carboxamide ribonucleoside facilitates tight junction assembly under conditions of normal extracellular Ca2+ concentrations and initiates tight junction assembly in the absence of Ca2+ as revealed by the relocation of zonula occludens 1, the establishment of transepithelial electrical resistance, and the paracellular flux assay. Expression of a dominant negative AMPK construct inhibits tight junction assembly in MDCK cells, and this defect in tight junction assembly can be partially ameliorated by rapamycin. These results suggest that AMPK plays a role in the regulation of tight junction assembly.