Water channel proteins (aquaporins) of the Plasma membrane Intrinsic Protein(PIP) subfamily provide means for fine and quick adjustments of the plant water status. A molecular model for gating of PIPs by cytosolic protons (H+) and divalent cations was derived from the atomic structure of spinach SoPIP2;1 in an open- and a closed-pore conformation. Here, we produced in Pichia pastoris the Arabidopsis AtPIP2;1 homolog, either wild-type (WT) or with mutations at residues supposedly involved in gating. Stopped-flow spectrophotometric measurements showed that, upon reconstitution in proteoliposomes, all forms function as water channels. First functional evidence for a direct gating of PIPs by divalent cations was obtained. In particular, cadmium and manganese were identified, in addition to calcium (Ca2+) and H+ as potent inhibitors of WT AtPIP2;1. Our data further show that His199, the previously identified site for H+ sensing, but also N-terminal located Glu31, and to a lesser extent Asp28, are involved in both divalent cations and H+-mediated gating. By contrast mutation of Arg124 rendered AtPIP2;1 largely insensitive to Ca2+ while remaining fully sensitive to H+. The role of these residues in binding divalent cations and/or stabilizing the open or closed pore conformations is discussed.