Human immunity to α(1,3)Galactosyl epitopes (αGal) may provide the means for a successful cancer gene therapy that uses the immune system to identify and to destroy tumor cells expressing the suicide gene α(1,3)Galactosyltransferase (αGT). Innate antibody specific for cell surface αGal constitutes a high percentage of circulating IgG and IgM immunoglobulins in humans and is the basis for complement-mediated hyperacute xenograft rejection and antibody-dependent cell-mediated cytotoxicity. In humans, the gene for αGT is mutated, and cells do not express the αGal moiety. We hypothesized that human tumor cells induced to express the αGal epitope would be killed by the hosts’ innate immunity. Previous in vitro work by our group has demonstrated complement-mediated lysis of αGal-transduced human tumor cells in culture by human serum. To induce antibodies to αGal in this in vivo study, αGT knockout mice were used to determine whether immunization with αGal could provide protection from challenge with αGal-expressing murine MC38 colon cancer cells. Knockout mice were immunized either a single time, or twice, with rabbit RBC. Antibody titers to αGal measured by indirect ELISA were significantly higher in mice immunized twice and approached the titers observed in human serum. Anti-αGal antibodies were predominantly of the IgG1 and IgG3 subtype. Immunized knockout mice were challenged i.p. with varying doses of αGal⁺ MC38 colon carcinoma cells. Nonimmunized control groups consisting of αGT knockout mice, and wild-type C57BL/6 mice were challenged as well with MC38 cells. Immunized mice survived and exhibited slower tumor development in comparison to nonimmunized knockout and control mice. This study demonstrates, in vivo, the protective benefit of an immune response to the αGal epitope. Our results provide a basis to pursue additional development of this cancer gene therapy strategy.