Brucellosis is an one of important zoonotic disease with enormous economic significance. Brucella causes infection in several animal species and humans. Prevention of the disease is necessary for eradication human and animal infection. Characterization and evaluation of different antigens of Brucella cells has a key role in progression of prevention programs. Here, we report the production and purification of recombinant 31kDa cell surface protein Brucella melitensis (BCSP31) and conjugated to detoxify LPS of Brucella melitensis for evaluation of its immunogenicity in BALA/c mice. In Tarbiat Modares University, Brucella 31kDa cell surface protein gene was cloned in pET28a (+) vector and expression in E coli BL21 (DE3) with 1mM IPTG and recombinant protein was purified by Ni-NTA agarose resins. Recombinant proteins were eluted with 250mM imidazol. Imidazol removed by dialysis. Proteins were assayed by Western-blotting and rBCSP31 was probed by Brucella rabbit anti serum. Purified protein injected to 32 semiannual BALB/c mice for survey of its protection effect. Percentage of clearance and log unit protection in injected mice showed the significant protection against colonization of Brucella melitensis in spleen of mice. BCSP31 were successfully cloned, expressed and purified. Injections of this recombinant protein can protection of mice against colonization in spleen versus of challenge strain.