Clinical studies reported hypomagnesaemia in long-term omeprazole usage that was probably due to intestinal Mg(2+) wasting. Our previous report demonstrated the inhibitory effect of omeprazole on passive Mg(2+) transport across Caco-2 monolayers. The present study aimed to identify the underlying mechanism of omeprazole suppression of passive Mg(2+) absorption. By using Caco-2 monolayers, we demonstrated a potent inhibitory effect of omeprazole on passive Mg(2+), but not Ca(2+), transport across Caco-2 monolayers. Omeprazole shifted the %maximum passive Mg(2+) transport-Mg(2+) concentration curves to the right, and increased the half maximal effective concentration of those dose-response curves, indicating a lower Mg(2+) affinity of the paracellular channel. By continually monitoring the apical pH, we showed that omeprazole suppressed apical acid accumulation. Neomycin and spermine had no effect on passive Mg(2+) transport of either control or omeprazole treated monolayers, indicating that omeprazole suppressed passive Mg(2+) transport in a calcium sensing receptor (CaSR)-independent manner. The results of western blot analysis showed that omeprazole significantly suppressed claudin (Cldn)-7 and -12, but not Cldn-2, expression in Caco-2 cells. By using apical solution of pH 5.5, 6.0, 6.5, and 7.0, we found that apical acidity markedly increased passive Mg(2+) transport, Mg(2+) affinity of the paracellular channel, and Cldn-7 and -12 expression in Caco-2 monolayers. Apical acidity abolished the inhibitory effect of omeprazole on passive Mg(2+) transport and Cldn-7 and -12 expression. Our results provided the evidence for the regulation of intestinal passive Mg(2+) absorption by luminal acidity-induced increase in Cldn-7 and -12 expression.