MicroRNAs (miRNAs) have emerged as key regulators in many biological processes, from development to defense, at almost all organismal levels. Recently, their role has been highlighted in pathogen-host interactions. Emerging evidence from a variety of virus-host systems indicates that cellular as well as virally encoded miRNAs influence viral replication. Here, we report changes in expression levels of host miRNAs upon ascovirus infection in an insect cell line and investigated the role of a host miRNA, Hz-miR24, in the host-virus system. We found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Experimental evidence demonstrated that Hz-miR24 downregulates ascoviral DNA-dependent RNA polymerase and its β subunit transcript levels late in infection. The specific miRNA-target interactions were investigated and confirmed using the ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Further, the expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. To our knowledge, this is the first cellular miRNA reported to interact with an insect virus.