In order to investigate the targeting relationship of miR-130c-5p to the potential target gene g in snakehead vesiculovirus (SHVV) infection and its effect on viral replication, the changes of viral gene and protein levels and miR-130c-5p in SHVV were determined in this study by quantitative real-time PCR (qRT-PCR) and Western blot techniques using channel catfish ovary (CCO) as experimental materials. In addition, the target sequence corresponding to miR-130c-5p on the g gene of SHVV was cloned into the plasmid pmirGLO, and the plasmid pmirGLO-G was constructed for dual luciferase reporter assay for target gene verification. The results showed that the expression levels of miR-130c-5p and g genes were significantly up-regulated with the increasing time and dose of SHVV infection. Further experiments showed that co-transfection of miR-130c-5p mimic and pmirGLO-G plasmid significantly inhibited luciferase activity, while transfection of miR-130c-5p inhibitor significantly up-regulated the fluorescence signal of pmirGLO-G reporter vector. In addition, overexpression of miR-130c-5p significantly reduced the mRNA and protein expression of the viral g gene, while inhibition of miR-130c-5p up-regulated the mRNA and protein expression levels of the g gene. The results showed that miR-130c-5p inhibited the proliferation of SHVV by targeting the g gene of SHVV and causing the degradation of G protein. The results of this study provide an important basis for understanding the pathogenic mechanism of microRNA regulation of SHVVV, and provide theoretical support for the development of anti-SHVV vaccines and other drugs.