MicroRNAs (miRNAs) are important regulators of biological processes, including host-virus interaction. This study investigated the involvement of Drosophila melanogaster miR-8-5p in host-virus interaction. Drosophila flies and cells challenged with Drosophila C virus (DCV) were found to have lower miR-8-5p abundance compared to uninfected samples. Lowering miR-8-5p abundance by experimental inhibition of the miRNA led to an increase in viral accumulation, suggesting that the observed decrease in the miR-8-5p abundance during DCV infection enhances viral replication. miR-8-5p putative targets were identified and included dJun, a transcription factor gene whose mammalian homologue cJun is induced by various viruses through kinase activation. Increasing miR-8-5p abundance using miR-8-5p mimics resulted in a decrease in dJun and GFP reporter levels. Furthermore, when the putative target in dJun was mutated, addition of miR-8-5p mimics did not result in the same antagonistic effect on dJun. These results show negative regulation of dJun by miR-8-5p and suggest that an miRNA-mediated pathway is involved in dJun regulation during viral infection. To analyse the role of dJun during DCV infection, dJun was knocked down in cells prior to DCV infection. Knockdown of dJun decreased DCV replication, providing evidence that dJun up-regulation that is concomitant with miR-8-5p down-regulation during DCV infection supports viral replication. These results highlight the role of miRNA in regulating the transcription factor gene dJun and uncover a previously unrecognized mechanism by which dJun is regulated during host-virus interaction.