Chiral pesticides with an asymmetrical center in their molecular structures possess enantioselectivity, not only in their pesticidal activities toward targeted organisms but also in toxicities to nontargeted organisms. Despite the fact that chiral pesticides deserve particular attention because of their ubiquitous presence in living and working environments, there has been limited research into their enantioselectivity in chronic toxicity. The immunotoxicity of chiral pesticides with respect to enantioselectivity has not been studied before. In this study, the role of enantioselectivity in the immunotoxicity of acetofenate (AF), an organochlorine insecticide, was investigated in an in vitro macrophage cell line model. Results of the cytotoxicity assay showed a clear dose-dependent growth inhibition effect of AF with enantioselectivity on RAW264.7 cells. S-(+)-AF was clearly more toxic to macrophages than R-(−)-AF and rac-AF. This work also demonstrated that S-(+)-AF possesses the strongest effects in induction of intracellular reactive oxygen species, DNA damage, and upregulation of p53 gene expression. These results, for the first time, show stark selectivity between enantiomers in their ability to induce macrophage-involved immunotoxicity of AF. These results suggest that assessment of the environmental safety and health risk of chiral contaminants should consider the role of enantioselectivity in immunotoxicity. In addition, our study will improve the knowledge of the role of enantioselectivity in immunotoxicity of chiral contaminants.