Eriochloa villosa (Thunb.) Kunth, the main weed in corn fields, has gradually developed resistance to nicosulfuron due to continuous and extensive application. We identified a biotype showing resistance to ALS inhibitor nicosulfuron with a resistant index 13.83, but without any target spot mutation. Herein, transcriptome sequencing was used to analyze the differences in gene expression at the transcriptional level between nicosulfuron-resistant E. villosa HEK-40 varieties and sensitive E. villosa HEK-15 varieties. The resistant and sensitive varieties comparison revealed 9931 DEGs after nicosulfuron application, of which 5426 and 4505 genes were up-regulated and down-regulated, respectively. Some contigs related to metabolic resistance were identified based on differential expression via RNA-Seq, which includes ABC transporters (ko02010), glucosinolate biosynthesis (ko00966), 2-oxocarboxylic acid metabolism (ko01210), alanine, aspartate, and glutamate metabolism pathways (ko00250). Seven CYP450 genes, four GST genes, ten ABC transporter genes, and two GT genes related to metabolic resistance were identified. The 10 candidate genes screened were validated using q-PCR. This validation indicates that activities associated with P450 enzymes, ABC transporters, and glutathione S-transferases (GST) may play a role in conferring resistance, which is important for reducing the impact of weeds on corn fields and ensuring food security.