Metolachlor, the chiral herbicide, inhibits the very-long-chain fatty acid (VLCFA) synthesis; elucidating the enantioselectivity between R-and S-metolachlor in the toxicological difference will facilitate the understanding of the site of action. We found that the endogenous accumulation of C22 VLCFAs decreased in both R-/S-metolachlor -treated plants by 6, 12, and 24 h after treatment, with more significant reduction in the S isomer group. Gene expression of glutathione S-transferase OsGST Tau members were obviously induced upon treatments with S or R isomer; both OsGSTU1 and OsGSTU4 can metabolize metolachlor effectively, with S isomer as the preference by directly catalyzing the conjugation between S-metolachlor and glutathione. In the current study, we provide the first evidence in rice seedlings that S-metolachlor showed herbicidal toxicity by blocking the synthesis of C22-type fatty acid, which eventually affects the whole elongation chain of (V)LCFA. Meanwhile, OsGSTU1 and 4 metabolize the metolachlor with the S isomer as preference. All of these discoveries broaden our knowledge about metolachlor toxicology and enantioselectivity.