Tight junction plays an important role in mediating paracellular permeability in epithelia. We previously found that activation of AMP-activated protein kinase (AMPK) increased saliva secretion by modulating paracellular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paracellular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance (TER), and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occludin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the interaction of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorylation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-modulated paracellular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in submandibular gland.