Abstract: 【Objective】 This study aimed to identify miRNAs involved in the physiological defense of Bemisia tabaci and investigate their influence on the control efficacy of Metarhizium anisopliae， to provide a theoretical basis for deve-loping nucleic acid pesticides and improving biological control strategies against Bemisia tabaci.【Method】 miRNA sequencing was performed to analyze differentially expressed miRNAs in Bemisia tabaci infected with Metarhizium anisopliae and to screen for those involved in physiological defense. Results were validated using real-time fluorescence quantitative PCR. Additionally， miRNA mimics were synthesized via a T7 in vitro transcription system and administered to Bemisia tabaci via feeding. The relative expression of these miRNAs and the survival rates of Bemisia tabaci following Metarhizium anisopliae infection were then measured.【Result】 Sequencing analysis revealed 179 differentially expressed miRNAs in infected Bemisia tabaci （56 up-regulated and 123 down-regulated）. Four miRNAs were selected for further study， including two novel miRNAs （Scaffold_40_27855 and Scaffold_1010_211） and two conserved miRNAs （gmo-miR-9-5p and age-miR-205）. Target prediction analysis indicated that these four miRNAs jointly targeted 56 functional genes involved in GO terms such as oxidoreductase activity， cAMP-dependent protein kinase complex， intracellular signal transduction， and carbohydrate transport， as well as KEGG pathways related to the immune system， digestive system， and environmental adaptation. Real-time fluorescence quantitative PCR confirmed that the relative expression of these four miRNAs was up-regulated in infected Bemisia tabaci. Furthermore， feeding Bemisia tabaci with the four miRNA mimics significantly up-regulated the corresponding miRNAs in vivo and decreased the survival rate of the Bemisia tabaci upon fungal infection.【Conclusion】 Metarhizium anisopliae infection induces the up-regulation of four specific miRNAs （Scaffold_40_27855， Scaffold_1010_211， gmo-miR-9-5p， and age-miR-205）. These miRNAs may facilitate fungal infection by inhibiting the expression of genes involved in the physiological defense of Bemisia tabaci. Consequently， the administration of miRNA mimics can enhance the virulence of Metarhizium anisopliae against Bemisia tabaci， thereby optimizing biocontrol efficacy.