This study was designed to investigate the inhibition activity of polysaccharide extract from Laminaria japonica against RSV. The polysaccharide from Laminaria japonica was isolated by ethanol precipitation. HEK293 cells were infected with RVS, and the antiviral activity of polysaccharide extract against RSV in host cells was tested. By using ELISA and western blot assay, the expression level of IFN-a and IRF3 and their functional roles in polysaccharide-mediated antiviral activity against RSV were investigated. The polysaccharide extract from Laminaria japonica had low toxicity to HEK293 cell. The TC50 to HEK293 cells was up to 1.76 mg/mL. Furthermore, the EC50 of polysaccharide extract to RSV was 5.27mg/mL, and TI was 334. The polysaccharide extract improved IRF-3 expression which promoted the level of IFN-a. In conclusion: Polysaccharide extract from Laminaria japonica elicits antiviral activity against RSV by upregulation of IRF3 signaling-mediated IFN-a production.