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Brown planthopper (BPH) is the most destructive insect pest to rice that causes tremendous yield loss each year in rice planting Asia and South-East Asia areas. Compared with traditional chemical-based treatment, utilization of plant endogenous resistance is a more effective and environmental-friendly way for BPH control. Accordingly, quite a few quantitative trait loci (QTLs) for BPH resistance were cloned using forward genetics. However, BPH is apt to change quickly into new biotypes to overcome plant resistance, therefore, new resistance resources and genes are continuously needed. miRNAs are important regulators in both plant development and physiological regulation including immunity, and might be used as effective supplements for BPH resistance QTLs. miR159 is an ancient and conserved miRNA. In this study, we found that each OsMIR159 gene in rice responded to BPH feeding very obviously, and genetic function assay proved them to negatively regulate BPH resistance, with STTM159 showing resistance to BPH, and over expression of OsmiR159d susceptible to BPH. One target genes of OsmiR159, OsGAMYBL2, positively regulated BPH resistance. Further biochemical studies revealed that OsGAMYBL2 could directly bind to the promoter of G-protein γ subunit encoding GS3 gene and repress its expression. And genetically, GS3 responded to BPH feeding promptly and negatively regulated BPH resistance, GS3 over expression plants were susceptible to BPH, while GS3 knock-out plants were resistant to BPH. Thus, we identified new function of OsmiR159-OsGAMYBL2 in mediating BPH response, and revealed a new OsmiR159-G protein pathway that mediates BPH resistance in rice., (© 2023. The Author(s).)