AbstractHerpesviruses have been reported to be able to encode and express functional viral microRNAs that target both viral and cellular transcripts. In our previous studies, we found a new miRNA miR‐KT‐635 encoded by Cyprinid herpesvirus 2, which is predicted to target viral genes and cellular genes involved in innate immune signalling pathway and apoptosis. However, the function and target gene of miR‐KT‐635 are not proved. In this study, the regulating target gene of miR‐KT‐635 was proved as the viral gene ORF23 directly, the target point sequence on gene was verified and miR‐KT‐635 was identified to regulate the expression of ORF23 protein. According to the bioinformatics analysis, the tRNA domain and ribosome domain in the protein sequence of ORF23 were found to share high homology with R2i and P53R2i, which are related to the ribonucleotide reductase small subunit in the host (transform NTP to dNTP). Within expectations, silencing of viral ORF23 or transfecting miR‐KT‐635 mimics in Carassius auratus gibelio caudal fin cell line (GiCF) could suppress viral propagation significantly.