Abstract: UDP-glycosyltransferases（UGTs） are important detoxification metabolizing enzymes in insects and play important roles in the development of insect resistance. A UDP-glucosyltransferase cDNA sequence from Mythimna separata was obtained by transcriptome sequencing and named UGT33J12（GenBank: MT873954） by UGT international nomenclature committee. The full-length cDNA sequence was 1 798 bp, containing an open reading frame of 1 557 bp, encoding a polypeptide of 518 amino acids with a molecular weight of 58.95 ku and an isoelectric point of 8.62. The N-terminal region of UGT33J12 gene contained a 20 amino acids signal peptide sequence, as well as histidine（H） and asparticacid（D）, which played a catalytic role, the C-terminal region contained two DBRs and a conserved characteristic motif, eight key amino acid residues were identified in the DBRs. The relative expression level of UGT33J12 was up-regulated when M. separata of the 4 th instar larvae was treated with lambda-cyhalothrin LD₁₀ for 12, 24 and 48 h, LD₃₀ and LD₅₀ for 12 and 24 h. The relative expression level of UGT33J12 was up-regulated when M. separata of the 4 th instar larvae was treated with chlorantraniliprole LD₁₀ for 24 and 48 h, LD₃₀ for 3, 12, 24 and 48 h, LD₅₀ for 3, 24 and 48 h. The results showed that UGT33J12 might be involved in the detoxification of lambda-cyhalothrin and chlorantraniliprole in M. separata.