Proton pump inhibitors (PPIs) are potent blockers of gastric acid secretion, used by millions of patients suffering from gastric acid-related complaints. Although PPIs have an excellent safety profile, an increasing number of case reports describe patients with severe hypomagnesemia due to long-term PPI use. As there is no evidence of a renal Mg2+ leak, PPI-induced hypomagnesemia is hypothesized to result from intestinal malabsorption of Mg2+. The aim of this study was to investigate the effect of PPIs on Mg2+ homeostasis in an in vivo mouse model. To this end, C57BL/6J mice were treated with omeprazole, under normal and low dietary Mg2+ availability. Omeprazole did not induce changes in serum Mg2+ levels (1.48 +/- 0.05 and 1.54 +/- 0.05 mmol/L in omeprazole-treated and control mice, respectively), urinary Mg2+ excretion (35 +/- 3 mumol/24 h and 30 +/- 4 mumol/24 h in omeprazole-treated and control mice, respectively), or fecal Mg2+ excretion (84 +/- 4 mumol/24 h and 76 +/- 4 mumol/24 h in omeprazole-treated and control mice, respectively) under any of the tested experimental conditions. However, omeprazole treatment did increase the mRNA expression level of the transient receptor potential melastatin 6 (TRPM6), the predominant intestinal Mg2+ channel, in the colon (167 +/- 15 and 100 +/- 7 % in omeprazole-treated and control mice, respectively, P < 0.05). In addition, the expression of the colonic H+,K+-ATPase (cHK-alpha), a homolog of the gastric H+,K+-ATPase that is the primary target of omeprazole, was also significantly increased (354 +/- 43 and 100 +/- 24 % in omeprazole-treated and control mice, respectively, P < 0.05). The expression levels of other magnesiotropic genes remained unchanged. Based on these findings, we hypothesize that omeprazole inhibits cHK-alpha activity, resulting in reduced extrusion of protons into the large intestine. Since TRPM6-mediated Mg2+ absorption is stimulated by extracellular protons, this would diminish the rate of intestinal Mg2+ absorption. The increase of TRPM6 expression in the colon may compensate for the reduced TRPM6 currents, thereby normalizing intestinal Mg2+ absorption during omeprazole treatment in C57BL/6J mice, explaining unchanged serum, urine, and fecal Mg2+ levels.