The use of inactivated influenza virus for the development of vaccines with broad heterosubtypic protection requires selective inactivation techniques that eliminate viral infectivity while preserving structural integrity. Here we tested if a hydrophobic inactivation approach reported for retroviruses could be applied to influenza. By this approach, the transmembrane domains of viral envelope proteins are selectively targeted by the hydrophobic photoactivatable compound, 1, 5 iodonaphthyl-azide (INA). This probe partitions into the lipid bilayer of the viral envelope and upon far UV irradiation reacts selectively with membrane-embedded domains of proteins and lipids while the protein domains that localize outside the bilayer remain unaffected. INA treatment of influenza virus blocked infection in a dose dependent manner without disrupting the virion or affecting neuraminidase activity. Moreover, the virus maintained the full activity in inducing pH dependent lipid mixing but pH dependent redistribution of viral envelope proteins into the target cell membrane was completely blocked. These results indicate that INA selectively blocks fusion of the virus with the target cell membrane at the pore formation and expansion step. Using a murine model of influenza virus infection, INA-inactivated influenza induced potent anti-influenza serum antibody and T cell responses, similar to live virus immunization, and protected against heterosubtypic challenge.INA treatment of influenza A virus produced a virus that is non-infectious, intact and fully maintains the functional activity associated with the ectodomains of its two major envelope proteins, neuraminidase and HA. When used as a vaccine given intranasally INA-influenza induced immune responses similar to live virus infection.