Vaccine-induced protection against infection by HIV or highly pathogenic and virulent SIV strains has been limited. In a proof-of-concept study, we show that a novel vaccine approach significantly protects rhesus macaques from mucosal infection by the highly pathogenic strain SIV mac251. We vaccinated three cohorts of 12 macaques each with live, irradiated vaccine cells secreting the modified endoplasmic reticulum chaperone gp96-Ig. Cohort 1 was vaccinated with cells secreting gp96 SIV Ig carrying SIV peptides. In addition, Cohort 2 received recombinant envelope protein SIV-gp120. Cohort 3 was injected with cells secreting gp96-Ig (no SIV Ags) vaccines. Cohort 2 was protected from infection. After seven rectal challenges with highly pathogenic SIV mac251, the hazard ratio was 0.27, corresponding to a highly significant, 73% reduced risk for viral acquisition. The apparent success of the novel vaccine modality recommends further study.