Myosins are key motor proteins of vertebrates that are important in cell-cell adhesion and migration; they are associated with viral infections. Herein, a myosin III light chain b gene from sea perch (Lateolabrax japonicus), designated Ljmyo3b, was characterized and its positive role in fish virus pathogenesis was revealed. The cDNA of Ljmyo3b encoded a 150-amino acid protein with two classical EF-hand domains; it shared the closest genetic relationship with Larimichthys crocea myo3b. Cellular distribution analysis showed that Ljmyo3b was localized in the cytoplasm and nucleus of LJB cells, and also on the cell surface, which was further confirmed by the protease protection assay. Spatial expression analysis revealed that Ljmyo3b was more abundant in the immune tissues of sea perch post red spotted grouper nervous necrosis virus (RGNNV) or viral hemorrhagic septicemia virus (VHSV) infection. In RGNNV- and VHSV-treated LJB cells, Ljmyo3b expression was significantly upregulated at 2, 4, 24, and 48 hpi. Furthermore, the ectopic expression of Ljmyo3b facilitated RGNNV and VHSV infection in LJB cells at 2, 4, and 24 hpi. Taken together, these results suggest that Ljmyo3b is involved in mediating RGNNV and VHSV infection in fish.