To investigate herbicide metabolism, human liver microsomes were incubated with threo- and erythro-isomers of flucetosulfuron. Each isomer produced one metabolite; the metabolites were unambiguously identified as enzymatic hydrolysis products by using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These metabolites were synthesized, producing white solids characterized using LC-MS/MS and nuclear magnetic resonance spectroscopy (1H and 13C). Using specific esterase inhibitors and activators, carboxylesterases and cholinesterases were demonstrated to be involved in flucetosulfuron metabolism. Under optimized metabolic conditions, the kinetic parameters for metabolite formation from threo-flucetosulfuron and erythro-flucetosulfuron were: Vmax, 151.41 and 134.38 nmol/min/mg protein, respectively; Km, 2957.37 and 2798.53 μM, respectively; and CLint, 51.20 and 48.02 μL/min/mg microsomes respectively. No significant kinetic differences were observed between the two isomers. These results indicated that the primary metabolic pathway for both flucetosulfuron isomers in human liver microsomes involves hydrolysis, catalyzed by carboxylesterase and cholinesterase.