AbstractMicropterus salmoides rhabdovirus (MSRV) is one of the common pathogens in the largemouth bass industry, which can cause lethal diseases in juvenile fish and enormous economic losses. To establish effective means to prevent MSRV infection, the pcDNA3.1‐G plasmid containing the MSRV glycoprotein gene was successfully constructed and intramuscularly injected into the largemouth bass to evaluate the immune responses and protective effects in our study. As the results showed, the serum antibody levels of the fish vaccinated with different doses of pcDNA3.1‐G were significantly higher compared with the control groups (PBS and pcDNA3.1). Meanwhile, the immune parameters (acid phosphatase and alkaline phosphatase) were also significantly up‐regulated. Several immune‐related genes (IgM, IL‐8, IL‐12p40 and CD40) were expressed in the pcDNA3.1‐G groups at higher levels than in the control groups, which indicated that strong immune responses were induced. Besides, the survival percentages of fish in the control groups (PBS and pcDNA3.1) and pcDNA3.1‐G groups (2.5, 5, 10 and 20 μg/fish) at 14 days after challenge experiment with MSRV were 0%, 0%, 6.1%, 15.2%, 29.0% and 48.5% respectively. This study indicated that pcDNA3.1‐G was a prospective DNA vaccine candidate against MSRV‐induced mortality.