miR-19-3p has been implicated in various pathological and physiological processes, including immune response, inflammation, oncogenesis and cell damage. However, its function in rainbow trout (Oncorhynchus mykiss) has not been well elucidated. In this study, the expression patterns of miR-19-3p and target gene DExH-Box helicase 58 (DHX58) in rainbow trout infected with infectious hematopoietic necrosis virus (IHNV) were detected, and regulatory mechanism and function of miR-19-3p were investigated by overexpression and inhibition experiment in vitro and in vivo. Expression patterns showed that miR-19-3p and DHX58 displayed significant time-dependent changes in IHNV-infected rainbow trout intestines, skins, gills, and liver cells, and their expression were negatively correlated at multiple time points. In vitro, the targeting relationship between miR-19-3p and DHX58 was confirmed by dual-luciferase reporter assay and RNA immunoprecipitation assay, and overexpression of miR-19-3p significantly suppressed the expression of DHX58 and downstream genes interferon regulatory factor 3 (IRF3), interferon regulatory factor 7 (IRF7), interferon (IFN), myxovirus 1 (MX1), interferon-stimulated gene 15 (ISG15), nuclear factor kappa-B (NF-κB), and interleukin-1 beta (IL-1β), whereas the expression levels of DHX58 and downstream genes were significantly increased after transfecting miR-19-3p inhibitor. In vivo, agomiR-19-3p significantly inhibited the expression of DHX58, and then reduced the expression levels of IRF3, IRF7, IFN, MX1, NF-κB, IL-1β, tumor necrosis factor-α (TNFα), and ISG15. Additionally, overexpression of miR-19-3p significantly increased IHNV copies and cell proliferation number, and suppressed apoptosis, while the opposite results were obtained after miR-19-3p repressing. This study confirmed that miR-19-3p regulates rainbow trout antiviral immune by DHX58-mediated interferon pathway in vitro and in vivo, which provides potential for using miRNAs as anti-viral target drugs.