A human influenza B/Lee/40 virus was cold-adapted by serial passages in embryonated chicken eggs, at progressively lower temperatures, for possible use as a future influenza B vaccine donor strain. Temperature sensitive and cold-adapted phenotypes were achieved as a consequence of the adaptation process. It was determined that the virus was attenuated in mice since the replication of the viral genome was significantly reduced in the lung. Despite decreased viral replication, the attenuated infection effectively induced a virus-specific immune response. We next developed a reassortant virus carrying two major surface proteins, hemagglutinin and neuraminidase from virulent B/Shangdong/7/97 and six internal genes from the cold-adapted B/Lee/40. The reassortant virus was also attenuated and protected mice from lethal challenge with wild type B/Shangdong/7/97. In addition, vaccination with the reassortant virus resulted in a specific antibody response and inhibited the replication of wild type virus in mice. We conclude that the cold-adapted B/Lee/40 donor strain merits further investigation as potential live vaccine carrier as an alternative means for protection from influenza B virus epidemics.