Aim: Clinical studies reported severe hypomagnesemia associated with long-term omeprazole usage, suggested the intestinal wasting. The present study aimed to elucidate the effect and underlying mechanism of omeprazole action on Mg2+ transport across intestinal epithelium. Methods: Caco-2 monolayer was cultured in culture media containing 200, 400, 600, 800, or 1,000 ng/nl omeprazole for 14 or 21 days before the measurement of bi-directional Mg2+ fluxes and electrical parameters in a modified Ussing chamber apparatus. Paracellular permeability of the monolayer was also determined by dilution potential technique and cation permeability study. The Arrhenius plot was performed to elucidate the activation energy of passive Mg2+ transport across the Caco-2 monolayers. The expression of claudi-2, -7, and -12 that highly expressed throughout small intestinal epithelium also observed. Results: Both apical to basolateral and basolateral to apical passive Mg2+ fluxes of omeprazoletreated epithelia were decreased in a dose- and time-dependent manner. Omeprazole also decreased the paracellular cation selectivity and changed the paracellular selective permeability profile of Caco-2 epithelium to Li+, Na+, K+, Rb+, and Cs+ from series VII to series VI of Eisenman sequence. The Arrhenius plot revealed a higher activation energy for passive Mg2+ transport in omeprazole-treated epithelium, indicating that omeprazole affected the paracellular channel of Caco-2 epithelium in such a way that Mg2+ movement was impeded. In addition, omeprazole significantly decreased claudin-7 and -12, but not -2, expression in Caco-2 cells. Conclusions: Omeprazole suppressed claudin-7 and -12 expresion. Omeprazole also decreased the paracellular cation permeability, possibly by decreased negative electrical field strength in the paracellular pores, and increased the activation energy for passive Mg2+ transport leading to the suppression of paracellular passive Mg2+ absorption. The present study provided the evidence about the adverse effect of omeprazole on intestinal Mg2+ absorption.