Brucella melitensis Rev.1 is the most effective vaccine against B. ovis infection in sheep but induces antibodies interfering with B. melitensis diagnosis. Brucella BP26 and Omp31 proteins are differential diagnostic antigens. Single or double bp26 and omp31 Rev.1 deletion mutants have been proven effective against B. melitensis in sheep. Here, the CGV26 (deleted in bp26 gene) and CGV2631 (deleted in both bp26 and omp31 genes) mutants have been tested for efficacy against B. ovis in rams. Either inoculated subcutaneously or conjunctivally, both mutants conferred significant protection against B. ovis. The protection induced by CGV26 was similar to that of Rev.1 but significantly higher than that conferred by CGV2631. In conclusion, the CGV26 mutant, in association with the adequate diagnostic strategy, could be a useful alternative to Rev.1 for sheep vaccination against B. ovis infections in those countries performing simultaneously B. melitensis and B. ovis eradication campaigns.