Vaccination against brucellosis using live attenuated strains is the primary approach in protecting livestock against the disease through a strong cellular immune response. Attenuated vaccine strains also induce serum anti-Brucella antibodies, mostly against Brucella O-polysaccharide, but their role in protection against the disease remains unclear. In this study, we show that Brucella OPS serum antibodies after vaccination or natural infection could kill Brucella in vitro as shown by the serum bactericidal activity (SBA) assay. We used serum samples of Rev. 1 vaccinated sheep that were negative or positive for Brucella OPS antibodies by either one of complement fixation test (CFT), microplate agglutination test (MAT) and ELISA, or sera of naturally infected sheep positive by CFT. We found a significant increase in the killing ability of sera 30 days after intraocular vaccination with Rev. 1 as compared with pre-vaccination. SBA was significantly higher in sera containing Brucella OPS IgG antibodies in comparison with sera lacking such antibodies (p < 0.001 against 16M & Rev. 1 strains). All 10 sera of convalescent sheep demonstrated significant killing ability against the 16M B. melitensis field strain. Specific OPS antibodies participate in the in vitro complement mediated Brucella killing suggesting a potential role in protection against the disease through this mechanism and relevance of developing OPS-based Brucella vaccines.