Abstract BACKGROUD MicroRNAs (miRNAs) are short single-stranded non-coding RNAs that regulate the expression of target genes, especially regulation or metabolism of endogenous or xenobiotic compounds. RESULTS The de novo assembly of the transcriptomes was obtained through Illumina short-read sequencing technology in Sitobion avenae. 57 miRNAs, of which 36 were known and 21 were novel were identified. Quantitative expression levels of miRNA showed that the expression of 5 miRNAs were significant up-regulation, and the expression of 11 miRNAs were significant down-regulation in the nymph of S. avenae treated by imidacloprid compared to the control, respectively. The putative transcript target genes in S. avenae that could be regulated by these miRNAs were also carried out. The potential functions of these miRNAs in the regulation of genes involved in the metabolism, regulatory or detoxification of S. avenae were clarified based on Gene Ontology and KEGG pathway. The effects of these miRNAs identified api-miR-1000, api-miR-316, and api-miR-iab-4 on susceptibility of S. avenae to imidacloprid was determined. Modulation of the abundance of api-miR-1000, api-miR-316, and api-miR-iab-4 through the addition of inhibitors of api-miR-1000, api-miR-316, and api-miR-iab-4 to the artificial diet significantly altered the susceptibility of S. avenae to imidacloprid, which further proved that the regulatory role of these miRNAs in regulation or metabolism of insecticides. CONCLUSION It suggested that differentially expressed microRNAs under the stress of imidacloprid could play critical regulatory role in the resistance of S. avenae to imidacloprid.