【Aim】 This study aimed to clone the chitin synthetase gene PxUAP and elucidate its function in Plutella xylostella. 【Method】 Based on the genomic information of diamondback moth, the full-length cDNA sequence of PxUAP was amplified using RT-PCR, and the expression pattern and function of the gene were analyzed using quantitative real-time PCR and RNA interference (RNAi). 【Result】 PxUAP (GenBank number: OR659549) contains an open reading frame of 1467 bp that encodes 488 amino acids with a predicted size of 54.985 ku and a theoretical isoelectric point of 6.12. Phylogenetic analysis demonstrated that the amino acid sequences of PxUAP and Papilio machaon (KPJ20218.1) UAP clustered into a single clade with 80.33% similarity. The developmental expression profile revealed that PxUAP was expressed at various developmental stages of P. xylostella, with the highest expression level in the male adult stage, and the tissue expression profile showed that the expression of PxUAP was highest in the legs and wings of female adults. After RNAi, the expression levels ofPxUAP and PxCHSA in the dsPxUAP injection group were significantly downregulated compared with those in the control group (dsGFP injection). At 24 and 48 h after RNAi, the expression level of PxCHSB was also significantly lower in the dsPxUAP treatment than in the dsGFP control group. In addition, most of P. xylostella could not pupate and died because of pupal shell shrinkage after RNAi, and the survival rate decreased to 0 after injection of dsPxUAP at 168 h. 【Conclusion】 PxUAP is a key regulatory gene in the chitin synthase pathway of diamondback moth and plays a crucial role in the growth and development of P. xylostella.