Acephate is a widely used organophosphorus insecticide globally, although there are some concerns about its usage with regard to acute consumer exposure and side-effects on nontarget organisms. These concerns are always attributed to the acephate metabolite methamidophos. In the many reports about the environmental behavior of acephate and its metabolite, none pay any attention to the chirality of them. In this study, the enantiomeric transformation and degradation of acephate was investigated in three soils under laboratory conditions using enantioselective GC-MS/MS. Racemic and enantiopure compounds were incubated in separate experiments. The degradation of racemates was shown to be enantioselective in unsterilized soils but not in the sterilized soils, thus confirming the enantioselectivity was microbially based. The priority of enantiomer degradation and transformation varied among soils and racemates. R-(+)-methamidophos was enriched in the Zhengzhou soil, but degraded faster in the Changchun and Nanchang soils than its antipode. For acephate, the Nanchang soil enriched R-(+)-acephate, and S-(-)-acephate accumulated in the other two soils. Acephate and methamidophos were both configurationally stable in soil, showing no interconversion of R-(+)- to S-(-)-enantiomers, or vice versa. The conversion of acephate to methamidophos proceeded with retention of configuration. Generally, the degradation followed approximate first-order kinetics, but showed significant lag phases.