Objective The purpose of this study was to analyze the immune responses induced by Brucellamelitensis(B. melitensis) outer membrane protein OMP25. Methods The omp25 gene from B. melitensis 16M was amplifiedby PCR. The fragment was cloned into pET-32a vector plasmid. The constructed recombinant plasmid pET32a-OMP25 wastransformed to E. coli BL21 (DE3) and was induced to express the fusion protein. Then the protein was purified using aNi2+ column purification kit. Mice were immunized with rOMP25 and current vaccine Rev. 1, and IFN-γ, IL-2 insplenocytes and IgG antibody and anti-inflammatory factor IL-10 levels in the serum were detected by ELISA. Thereactionogenicity was detected by Western Blot. Results (1) The full length of the omp25 gene was 642 bp, encoding 214amino acids. rOMP25 was approximately 40. 8 × 10³ as measured by SDS-PAGE. There was a single band after purification. (2) The t-test method showed that mice were immunized with rOMP25 and Rev. 1, the levels of IFN-γ andIL-2 in splenocytes, IgG antibody and IL-10 levels in serum in the rOMP25 group were similar to those in the Rev. 1 group,and significantly greater than those in the control ( P < 0. 01). (3) rOMP25 had good reactivity as observed in the WestemBlot. These result confirmed that rOMP25 could induce the body to produce high levels of cellular and humoral immunity,with good reactivity. Conclusions This study provides technical support for further study of the function of OMP25 proteinfor B. melitensis related vaccine development.