The recombinant canarypox vector, ALVAC-HIV, together with human immunodeficiency virus (HIV) gp120 envelope glycoprotein, has protected 31.2% of Thai individuals from HIV acquisition in the RV144 HIV vaccine trial. This outcome was unexpected, given the limited ability of the vaccine components to induce CD8⁺ T-cell responses or broadly neutralizing antibodies. We vaccinated macaques with an immunization regimen intended to mimic the RV144 trial and exposed them intrarectally to a dose of the simian immunodeficiency virus SIV_mac251 that transmits few virus variants, similar to HIV transmission to humans. Vaccination induced anti-envelope antibodies in all vaccinees and CD4⁺ and CD8⁺ T-cell responses. Three of the 11 macaques vaccinated with ALVAC-SIV/gp120 were protected from SIV_mac251 acquisition, but the result was not significant. The remaining vaccinees were infected and progressed to disease. The magnitudes of vaccine-induced SIV_mac251-specific T-cell responses and binding antibodies were not significantly different between protected and infected animals. However, sera from protected animals had higher avidity antibodies to gp120, recognized the variable envelope regions V1/V2, and reduced SIV_mac251 infectivity in cells that express high levels of α₄β₇ integrins, suggesting a functional role of antibodies to V2. The current results emphasize the utility of determining the titer of repeated mucosal challenge in the preclinical evaluation of HIV vaccines.