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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-<i>Brucella</i> antibodies, mostly against <i>Brucella</i> O-polysaccharide, but their role in protection against the disease remains unclear. In this study, we show that <i>Brucella</i> OPS serum antibodies after vaccination or natural infection could kill <i>Brucella</i> 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 <i>Brucella</i> 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 <i>Brucella</i> OPS IgG antibodies in comparison with sera lacking such antibodies (<i>p</i> < 0.001 against 16M & Rev. 1 strains). All 10 sera of convalescent sheep demonstrated significant killing ability against the 16M <i>B. melitensis</i> field strain. Specific OPS antibodies participate in the in vitro complement mediated <i>Brucella</i> killing suggesting a potential role in protection against the disease through this mechanism and relevance of developing OPS-based <i>Brucella</i> vaccines.