BACKGROUND: S-metolachlor (MET) was used to prevent weed infestation in sorghum fields, but inappropriate application could result in phytotoxicity on sorghum. Exogenous gibberellin A3 (GA3 ) has been applied for alleviating the phytotoxicity of MET. However, its detoxification mechanism is still not well known.RESULTS: Leaf deformity of sorghum caused by 200 mg/L MET was alleviated by treating sorghum shoots with 800 mg/L GA3 , and the injury recovery rate of growth index was over 73%. More importantly, GA3 could not accelerate the metabolic rate of MET in sorghum. The result of phytohormone metabolomics showed that endogenous GA3 content in sorghum decreased by 78.10% with MET treatment, while abscisic acid (ABA) content increased by 120.2%, resulting in 10.3-fold increase of ABA/GA3 ratio. Content of ABA and GA3 increased by 11.9- and 21.1-fold with MET and GA3 treatment, respectively, leading to ABA/GA3 ratio restoration. Moreover, MET inhibited the expression of genes encoding key enzymes related to GA synthesis including CPS1, KO2, KAO, GA20ox1D and ABA8ox gene related to ABA metabolism. The transcription levels of GA metabolism-related genes CYP714D1 and GA2ox were up-regulated by 11.2- and 7.2-fold, while ABA synthesis-related genes NCED and ZEP were up-regulated by 8.0- and 3.0-fold, respectively, with MET and GA3 treatment.CONCLUSION: In this study, exogenous GA3 protecting sorghum shoots from MET phytotoxicity was due to supplement the MET-induced GA3 deficiency by absorbing exogenous GA3 , and restore homeostasis of ABA and GA3 by promoting ABA synthesis, which provides novel insights for mechanism of GA3 alleviating MET phytotoxicity. © 2022 Society of Chemical Industry.