The host cell protease TMPRSS2 cleaves the influenza A virus (IAV) hemagglutinin (HA). Several reports have described resistance of Tmprss2-/- knock-out (KO) mice to IAV infection but IAV of the H2 subtype have not been examined yet. Here, we demonstrate that TMPRSS2 is able to cleave H2-HA in cell culture and that Tmprss2-/- mice are resistant to infection with a re-assorted PR8_HA(H2) virus. Infection of KO mice did not cause major body weight loss or death. Furthermore, no significant increase in lung weights and no virus replication were observed in Tmprss2-/- mice. Finally, only minor tissue damage and infiltration of immune cells were detected and no virus-positive cells were found in histological sections of Tmprss2-/- mice. In summary, our studies indicate that TMPRSS2 is required for H2 IAV spread and pathogenesis in mice. These findings extend previous results pointing towards a central role of TMPRSS2 in IAV infection and validate host proteases as a potential target for antiviral therapy.