V-type nerve agents, known as VX, are organophosphate (OP) compounds, and show extremely toxic effects on human and animals by causing cholinergic overstimulation of synapses. The bacterial organophosphorus hydrolase (OPH) has attracted much attention for detoxifying V-type agents through hydrolysis of the P-S bond. However, low catalytic efficiency of OPH has limited the practical use of the enzyme. Here we present rational design of OPH with high catalytic efficiency for a V-type nerve agent. Based on the model structure of the enzyme and substrate docking simulation, we predicted the key residues that appear to enhance the access of the substrate to the active site of the enzyme, and constructed numerous OPH mutants. Of them, double mutant, L271/Y309A, was shown to exhibit a 150-fold higher catalytic efficiency for VX than the wild-type.