The recent outbreaks of highly pathogenic avian influenza in Asia and spread of the disease worldwide highlight the need to redefine conventional immunization approaches and establish effective mass vaccination strategies to face global pandemics. Transcutaneous immunization (TCI) is a novel route for vaccination, which uses the topical application of vaccine antigens on the skin. In this study, we investigated the potential of TCI using inactivated whole influenza virus. We found that TCI with whole inactivated influenza virus induced influenza virus-specific antibodies with hemagglutination inhibition and neutralizing activities as well as cellular immune responses, even without an adjuvant, and conferred protective immunity to virus challenge. Co-administration with cholera toxin (CT), a potent adjuvant for TCI, significantly enhanced immune responses against the influenza virus antigen. To enhance penetration of the skin barrier to the particulate influenza viral antigens, we tested the effects of the potential penetration enhancers/immunomodulators oleic acid (OA) and retinoic acid (RA). Pretreatment of mouse skin with OA elicited increased levels of influenza virus-specific binding and neutralizing antibodies to levels equivalent to those induced by intranasal immunization with inactivated influenza virus. OA and RA treatments differentially affected the pattern of cytokine production upon stimulation with influenza viral antigen and provided enhanced protection. These results reveal a promising perspective for the application of transcutaneous immunization to prevent influenza epidemics as well as a range of other infectious diseases.