Abstract α-Galactosylceramide (α-GalCer), originally isolated from a marine sponge, was known to activate natural killer T (NKT) cells through CD1d-mediated Ag presentation and induce Th1 and/or Th2 immunity. In this study, we evaluated the nasal adjuvanticity of α-GalCer when co-administered with formalin-inactivated influenza virus A/PR/8/34 (PR8) in BALB/c mice. A single nasal immunization of inactivated PR8 and α-GalCer induced brisk levels of PR8-specific IgG and IgA Abs in serum and lung washes. Antigen-specific Ab responses lasted for 3 months, providing protective immunity against challenge with live PR8. In addition, mice given α-GalCer also exhibited cellular immune responses including cytotoxic T lymphocyte (CTL) generation. Because it did not redirect Ags into brain, α-GalCer would likely pose no risk if administered as a nasal adjuvant. These results suggest for the first time that a single nasal immunization of inactivated virus and α-GalCer is a safe and effective means of preventing influenza infection.