Human serum paraoxonase 1 (HuPON1; EC 3.1.8.1) is a calcium‐dependent six‐fold β‐propeller enzyme that has been shown to hydrolyze an array of substrates, including organophosphorus (OP) chemical warfare nerve agents. Although recent efforts utilizing site‐directed mutagenesis have demonstrated specific residues (such as Phe222 and His115) to be important in determining the specificity of OP substrate binding and hydrolysis, little effort has focused on the substrate stereospecificity of the enzyme; different stereoisomers of OPs can differ in their toxicity by several orders of magnitude. For example, the C±P– isomers of the chemical warfare agent soman (GD) are known to be more toxic by three orders of magnitude. In this study, the catalytic activity of HuPON1 towards each of the four chiral isomers of GD was measured simultaneously via chiral GC/MS. The catalytic efficiency (kcat/Km) of the wild‐type enzyme for the various stereoisomers was determined by a simultaneous solution of hydrolysis kinetics for each isomer. Derived kcat/Km values ranged from 625 to 4130 mm−1·min−1, with isomers being hydrolyzed in the order of preference C+P+ > C–P+ > C+P– > C–P–. The results indicate that HuPON1 hydrolysis of GD is stereoselective; substrate stereospecificity should be considered in future efforts to enhance the OPase activity of this and other candidate bioscavenger enzymes.