Influenza B viruses (IBV) are transmitted among humans, with disease being worse in men than women. C57BL/6 male and female mice were inoculated with Victoria lineage B/Brisbane/60/2008 containing a PB2 F406Y mutation. Juvenile, adult, and aged males exhibited greater virus titers, morbidity, and pulmonary inflammation than age-matched females. Oseltamivir treatment reduced virus titers in males thereby reducing morbidity and pulmonary cytokine responses to female-equivalent levels. Infection of transgenic and mutant mice that allowed for separation of sex chromosome dosage from gonadal sex effects revealed that the presence of a Y chromosome (ChrY) was the major contributing factor for male-biased susceptibility to IBV. IBV infection suppressed pulmonary Uty and Ddx3y expression in ChrY-bearing mice, which was reversed by oseltamivir treatment, suggesting that virus replication inhibits protective ChrY gene expression, causing male-biased IBV pathogenesis.