The green foxtail, (L.) P. Beauv. (Poales: Poaceae), is a troublesome and widespread grass weed in China. The acetolactate synthase (ALS)-inhibiting herbicide nicosulfuron has been intensively used to manage , and this has substantially increased the selection pressure. Here we confirmed a 35.8-fold resistance to nicosulfuron in an population (R376 population) from China and characterized the resistance mechanism. Molecular analyses revealed an Asp-376-Glu mutation of the gene in the R376 population. The participation of metabolic resistance in the R376 population was proved by cytochrome P450 monooxygenases (P450) inhibitor pre-treatment and metabolism experiments. To further elucidate the mechanism of metabolic resistance, eighteen genes that could be related to the metabolism of nicosulfuron were obtained bythe RNA sequencing. The results of quantitative real-time PCR validation indicated that three ATP-binding cassette (ABC) transporters (, , and ), four P450 (, , , and ), and two UDP-glucosyltransferase (UGT) ( and ), and one glutathione S-transferases (GST) () were the major candidates that contributed to metabolic nicosulfuron resistance in . However, the specific role of these ten genes in metabolic resistance requires more research. Collectively, gene mutations and enhanced metabolism may be responsible for the resistance of R376 to nicosulfuron.