Lithium promotes the phosphorylation of glycogen synthase kinase-3β (GSK3β), and this reaction protects against acute kidney injury mediated by renal apoptosis. Lithium is considered to be reabsorbed by sodium-phosphate cotransporters and sodium-proton exchanger NHE3. This study evaluated the relation between the lithium reabsorption and the phosphorylation of GSK3β, by using acetazolamide, an NHE3 inhibitor. In rats infused with lithium chloride, the plasma concentration of lithium was 4.77 mEq/l, and the renal clearance of lithium and its fractional excretion were calculated to be 2.29 ml/min/kg and 0.405, respectively. Coadministration of acetazolamide decreased creatinine clearance and the reabsorption rate of lithium, increased the fractional excretion of lithium, and did not affect its plasma concentration. Western blot analysis exhibited the facilitation of GSK3β phosphorylation in the kidney cortex by lithium infusion, and acetazolamide inhibited the lithium-induced phosphorylation of GSK3β. Lithium did not affect GSK3β phosphorylation in the liver and did not affect Akt in the kidney cortex and liver. These data show that lithium reabsorption contributes to GSK3β phosphorylation in the kidney cortex.