AbstractCuticular resistance, characterized by decreased epidermal penetration, has been reported on highly pyrethroid‐resistant mosquitoes. In this study, we examined the role of genes in the chitin biosynthetic pathway in the context of deltamethrin‐resistant (DR) Culex pipiens pallens. We found that expression of the trehalase (TRE1) gene and chitin synthase (CHS1) gene was upregulated 1.65‐ and 1.75‐fold with quantitative reverse transcription polymerase chain reaction, respectively, in the DR strain as compared with the deltamethrin‐susceptible (DS) strain. Examination of chitin content in DR and DS pupae showed an increased amount of chitin in DR pupae. To further establish the role of TRE1 and CHS1 in deltamethrin resistance, we injected mosquitoes with small interfering RNA (siRNA) for knockdown of TRE1 or CHS1 expression. The mortality rates of DR mosquitoes exposed to insecticides increased 17% and 26% after siTRE1 and siCHS1 injection, respectively. The siRNA treatment against TRE1 resulted in decreased expression of the downstream gene CHS1. Together, our findings support a role of TRE1 and CHS1 in the regulation of pyrethroid resistance.