Influenza B virus is a human pathogen whose origin and possible reservoir in nature are not known. An influenza B virus was isolated from a naturally infected harbor seal (Phoca vitulina) and was found to be infectious to seal kidney cells in vitro. Sequence analyses and serology indicated that influenza virus B/Seal/Netherlands/1/99 is closely related to strains that circulated in humans 4 to 5 years earlier. Retrospective analyses of sera collected from 971 seals showed a prevalence of antibodies to influenza B virus in 2% of the animals after 1995 and in none before 1995. This animal reservoir, harboring influenza B viruses that have circulated in the past, may pose a direct threat to humans.