The functions of the Betanodavirus non-structural protein B1 is still unknown. We examined B1 expression patterns and investigated novel cell death regulatory functions for this viral protein following RGNNV infection in fish cells. The B1 gene (336 nt) was cloned from the redspotted grouper nervous necrosis virus (RGNNV) genome. B1 mRNA was rapidly expressed in the fish cells from viral RNA3 at 12 h post-infection (p.i.). At the protein level, expression was low at 12 h p.i., and then increased rapidly between 24 h and 72 h p.i. In RGNNV-infected, B1-containing fish cells, over expression of RGNNV B1 reduced Annexin-V positive cells by 50% and 65% at 48 h and 72 h p.i., respectively, and decreased loss of mitochondrial membrane potential (MMP) by 20% and 70% at 48 h and 72 h p.i., respectively. Finally, B1 knockdown during RGNNV infection using anti-sense RNA increased necrotic cell death and reduced cell viability during the early replication cycle (24 h p.i.). Our results suggest that B1 is an early expression protein that has an anti-necrotic cell death function which reduces the MMP loss and enhances viral host cell viability. This finding provides new insights into RNA viral pathogenesis and disease control.