The extensive use of herbicides has raised concerns about crop damage, necessitating the development of effective herbicide safeners. Fluxofenim has emerged as a promising herbicide safener; however, it's underlying mechanism remains unclear. Here, we screened two inbred lines 407B and HYZ to investigate the detoxication of fluxofenim in mitigating metolachlor damage in sorghum. Metolachlor inhibited seedling growth in both 407B and HYZ, while, fluxofenim could significantly restore the growth of 407B, but not effectively complement the growth of HYZ. Fluxofenim significantly increased the activities of glutathione-S-transferase (GST) to decrease metolachlor residue in 407B, but not in HYZ. This implys that fluxofenim may reduce metolachlor toxicity by regulating its metabolism. Furthermore, metolachlor suppressed AUX-related and JA-related genes expression, while up-regulated the expression of SA-related genes. Fluxofenim also restored the expression of AUX-related and JA-related genes inhibited by metolachlor and further increased expression of SA-related genes. Moreover, we noted a significant increase in the content of trans-zeatin O-glucoside (tZOG) and Gibberellin1 (GA1) after the fluxofenim treatment. In conclusion, fluxofenim may reduce the injury of herbicide by affecting herbicide metabolism and regulating hormone signaling pathway.