Aquaporin-2 (AQP2) is a water channel protein expressed in principal cells (PCs) of the kidney collecting ducts (CDs) and plays a critical role in mediating water reabsorption and urine concentration. AQP2 undergoes both regulated trafficking mediated by vasopressin (VP) and constitutive recycling, which is independent of VP. For both pathways, actin cytoskeletal dynamics is a key determinant of AQP2 trafficking. We report here that manganese chloride (MnCl₂) is a novel and potent regulator of AQP2 trafficking in cultured cells and in the kidney. MnCl₂ treatment promoted internalization and intracellular accumulation of AQP2. The effect of MnCl₂ on the intracellular accumulation of AQP2 was associated with activation of RhoA and actin polymerization without modification of AQP2 phosphorylation. Although the level of total and phosphorylated AQP2 did not change, MnCl₂ treatment impeded VP-induced phosphorylation of AQP2 at its serine residues 256, 264, and 269 and dephosphorylation at serine 261. In addition, MnCl₂ significantly promoted F-actin polymerization along with downregulation of RhoA activity, and prevented VP-induced membrane accumulation of AQP2. Finally, MnCl₂ treatment in mice resulted in significant polyuria and reduced urinary concentration, likely due to intracellular relocation of AQP2 in the PCs of kidney CDs. More importantly, the reduced urinary concentration caused by MnCl₂ treatment in animals was not corrected by VP. In summary, our study identified a novel effect of MnCl₂ on AQP2 trafficking through modifying RhoA activity and actin polymerization, and uncovered its potent impact on water diuresis in vivo.