Spodoptera frugiperda, commonly known as the fall armyworm (FAW), is a major pest across the globe due to its broad host range and distribution worldwide. We investigated the function of microRNAs (miRNAs) in the detoxification of insecticides, with a specific focus on its susceptibility to chlorantraniliprole which is widely utilized insecticide for its management. miRNAs are small non-coding RNA molecules, crucial for post-transcriptional regulation of gene expression. This study aims to elucidate the impact of these miRNAs on the expression of cytochrome P450 genes, which play a significant role in conferring insecticide resistance. We identified notable changes in the abundance of two specific miRNAs, sfr-miR-10465-5p and sfr-miR- 10476-5p through RNA sequencing, after chlorantraniliprole exposure. These miRNAs exhibited significantly high expression in the fat body tissue, while showing relatively lower expression in the head, midgut, and malpighian tubules. Further analysis suggested that these miRNAs might target specific cytochrome P450 genes, like CYP4C1 and CYP4C21, which are known to play a role in insecticide resistance development. Experimentation with miRNA mimics through microinjection revealed a notable increase in the survival rates of S. frugiperda larvae when subjected to chlorantraniliprole exposure, with a significant reduction in CYP4C1 and CYP4C21 gene expression levels. This suggests a direct connection between the miRNAs and the increased tolerance of Spodoptera larvae to the insecticide. Our research presents the complex function of miRNAs in gene expression regulation related to insecticide resistance, offering valuable insights into the molecular mechanisms of chlorantraniliprole resistance in S. frugiperda. These findings pave the way for further investigations into miRNA roles and their potential in managing pesticide resistance in agricultural pests.