Background: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. Objective: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. Methods: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. Results: It was determined that doses of the recombinant Omp25 protein doses greater than 0.1 mu g/mL are toxic to RAW cells. Doses of 1 mu g/mL and lower significantly increased inflammation due to nitric oxide (NO) levels. ELISA results showed that IFN-gamma was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 mu g/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. Conclusion: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.