Duck Tembusu Virus (DTMUV), belonging to the Flaviviridae family. Ducks of different breeds and ages can be naturally infected with DTMUV. MicroRNAs (miRNAs) play a important role in viral replication. However, little is known about the function of miRNAs during DTMUV infection. Here, the host miR-146b-5p was found to regulate DTMUV replication. When DTMUV infected duck embryo fibroblasts (DEFs), the expression levels of miR-146b-5p increased significantly over time. Moreover, the viral RNA copies, E protein expression levels and virus titers were all upregulated when miR-146b-5p was overexpressed in DEFs. The opposite results were also observed upon knockdown of miR-146b-5p in DEFs. To explore the mechanism by which miR-146b-5p promoted DTMUV replication, mass spectrometry and RNA pull-down assays were employed. Ribosomal protein S14 (RPS14), was identified to interact with miR-146b-5p. In addition, the relative mRNA expression levels of RPS14 gene were negatively modulated by miR-146b-5p. Subsequently, it was further found that miR-146b-5p promoted DTMUV replication by targeting RPS14. This study revealed provides a new mechanism by which DTMUV evades host defenses and a new direction for further antiviral strategies development. https://doi.org/10.1016/j.psj.2023.102890.