Diclofop-methyl (DM) is a widely used chiral herbicide, which rapidly hydrolyzes to its major metabolite diclofop acid (DC) after application. With a carbon chiral center, DC not only is an important ingredient of herbicidal activity, but also has a long half-life in soil. Studies so far have only considered the activity of racemic DM in target organisms, and the enantioselective toxicity in nontarget plants of DM and DC has yet to be explored. In this study, the enantioselective phytotoxicity of DC mediated by oxidative stress and the key enzyme ACCase in the fatty acid synthesis system on the model plant Arabidopsis thaliana was investigated. Significant differences between the two enantiomers were observed in phytotoxicity including growth inhibition, oxidative damage and alteration of key genes expression of ACCase, with R-DC showing greater toxicity to Arabidopsis thaliana than S-DC. The results of molecular docking showed that there was a stronger affinity between R-DC and the target enzyme carboxyltransferase domain of ACCase, likely leading to the enantioselective phytotoxicity of DC. This study suggested that chirality of both parent compounds and metabolites should be considered to improve our understanding of the environmental fate and risks of chiral pesticides.