Spring Viremia of Carp Virus (SVCV) is a highly pathogenic pathogen that infects carp fish, and SVCV infection frequently causes a severe inflammatory response in the affected fish. The viral infection is extremely lethal in carp larvae, often leading to heavy economic losses. Cholic acid (CA) is a primary bile acid synthesized directly from cholesterol in the liver and has been found to have an inhibitory effect on viral infections. To substantiate this antiviral potential, 500 μM CA was tested against SVCV replication in vitro and showed a significant inhibitory effect on viral replication. Further experimental tests demonstrated that CA exerted antiviral effects during its replication phase but not at viral adsorption and entry stages. In vivo experiments demonstrated that CA increased the survival rate of SVCV-infected zebrafish by 40 %, reduced viral load in tissues and reduced histopathological lesions. Comparison of the liver transcriptome of zebrafish between CA treatment and control (NC) groups revealed that CA treatment was able to promote the synthesis of polyunsaturated fatty acids, with the most significant changes in docosahexaenoic acid (DHA). Inhibition assay on fatty acid desaturase 2 (Fads2), the first rate-limiting enzyme for the synthesis of polyunsaturated fatty acid, showed that the antiviral effect of CA on SVCV disappeared. These new findings suggest that CA may exert its antiviral effects by regulating the synthesis of polyunsaturated fatty acid. In addition, this study also verified that DHA has antiviral activity against SVCV. In conclusion, CA shows promise as a potential anti-SVCV drug or feed additive that could help mitigate SVCV outbreaks in aquaculture.