Recently, the development of antiviral agents with novel mechanisms of action has been required since many types of infectious disease have become a serious problem in our society. In the present study, we isolated a novel acidic polysaccharide, nostoflan (NSF), from a terrestrial blue-green alga, Nostoc flagelliforme, and examined its structure and antiviral activity. The sugar composition and methylation analyses of NSF revealed that it is mainly composed of (-->4)- D-Glcp-(1-->, -->6,4)-D-Glcp-(1-->, -->4)-D-Galp-(1-->, -->4)-D-Xylp-(1-->, D-GlcAp-(1-->, D-Manp-(1-->) with a ratio of ca. 1:1:1:1:0.8:0.2. Oligosaccharide analysis after partial acid hydrolysis of NSF revealed that this polysaccharide might be mainly composed of the sugar sequences of (-->4)-beta-D-Glcp-(1-->4)-D-Xylp-(1 and-->4)-[beta-D-GlcAp-(1-->6)-]-beta-D-Glcp-(1-->4)-D-Galp-(1-->). NSF showed potent antiviral activities against several enveloped viruses including herpes simplex virus type 1, type 2 (HSV-1, HSV-2), human cytomegalovirus, and influenza A virus (IFV). NSF selectively inhibited the attachment of HSV-1 to host cells but not its penetration phase. In an experimental animal study where IFV-infected mice received NSF intranasally, the mortality of mice was significantly decreased. Neutralizing titers in sera of mice treated with NSF were higher than in those treated with oseltamivir. From these results, NSF was found to be a novel polysaccharide that shows antiviral activity in vitro and in vivo in spite of a nonsulfated polysaccharide.