The HIV-1 Tat protein plays a critical role in the pathogenesis of HIV and has been considered as a candidate vaccine antigen. In an effort to design a non-invasive vaccination strategy against HIV-1 that stimulates the induction of systemic and mucosal immune responses, we studied the transcutaneous delivery of a synthetic Tat protein using cholera toxin as an adjuvant. Following immunization of BALB/c mice with various doses of Tat, IgG and IgA antibody responses were measured in the serum and vaginal washes, respectively. Serum antibodies predominantly recognized the N-terminal and basic functional domains of the protein and exhibited neutralizing capacity against Tat-driven transactivation. Transcutaneous immunization also elicited potent cellular immune responses against Tat and the secretion of high levels of IL-2, IFN-gamma and IL-6. These findings demonstrate for the first time that by using a simple and safe immunization procedure, a synthetic Tat protein can elicit potentially protective immune responses. Transcutaneous immunization may be advantageous for the non-invasive delivery of other HIV candidate vaccine antigens.