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The RNA interference pathway mediated by microRNAs (miRNAs) is one of the methods to defend against viruses in insects. Recent studies showed that miRNAs participate in viral infection by binding to target genes to regulate their expression. Here, we found that the Bombyx mori miRNA, miR‐6498‐5p was down‐regulated, whereas its predicted target gene pyridoxal phosphate phosphatase PHOSPHO2 (BmPLPP2) was up‐regulated upon Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Both in vivo and in vitro experiments showed that miR‐6498‐5p targets BmPLPP2 and suppresses its expression. Furthermore, we found miR‐6498‐5p inhibits BmNPV genomic DNA (gDNA) replication, whereas BmPLPP2 promotes BmNPV gDNA replication. As a pyridoxal phosphate (PLP) phosphatase (PLPP), the overexpression of BmPLPP2 results in a reduction of PLP content, whereas the knockdown of BmPLPP2 leads to an increase in PLP content. In addition, exogenous PLP suppresses the replication of BmNPV gDNA; in contrast, the PLP inhibitor 4‐deoxypyridoxine facilitates BmNPV gDNA replication. Taken together, we concluded that miR‐6498‐5p has a potential anti‐BmNPV role by down‐regulating BmPLPP2 to modulate PLP content, but BmNPV induces miR‐6498‐5p down‐regulation to promote its proliferation. Our findings provide valuable insights into the role of host miRNA in B. mori–BmNPV interaction. Furthermore, the identification of the antiviral molecule PLP offers a novel perspective on strategies for preventing and managing viral infection in sericulture. [ABSTRACT FROM AUTHOR]