The spinach plant aquaporin SoPIP2;1 is a gated water channel, which switches between open and closed states depending on the conformation of a 20-residue cytoplasmic loop, the D-loop. Using fully atomistic molecular dynamics simulations, we have investigated the possibility of driving the conformational equilibrium of the protein toward a constitutively open state. We introduce two separate mutations in the D-loop, while being in the closed conformation. We show that the single channel permeability of both mutants is comparable to that of the open conformation. This Article provides new molecular insight into the gating mechanism of SoPIP2;1. It is proposed that residues Arg190, Asp191, and Ser36 might play important roles in the gating of the protein.