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Abstract Brucellosis is an infectious disease that brings enormous economic burdens for developing countries. The Brucella melitensis (B. melitensis) M5-90 vaccine strain (M5-90) has been used on a large scale in China, but may cause abortions if given to pregnant goats or sheep subcutaneously during the late stages of gestation. Moreover, the vaccine M5-90 cannot differentiate natural from vaccinated infection. Therefore, a safer and more potent M5-90 vaccine is required. In this study, a vjbR mutant of M5-90 (M5-90Δ vjbR) was constructed and overcame these drawbacks. M5-90Δ vjbR strain showed reduced survival capability in murine macrophages (RAW 264.7) and BALB/c mice and induced high protective immunity in mice. In addition, M5-90Δ vjbR induced an anti- Brucella -specific immunoglobulin G (IgG) response and stimulated the expression of gamma interferon (INF-γ) and interleukin-4 (IL-4) in vaccinated mice. Furthermore, M5-90Δ vjbR induced IgG response and stimulated the secretion of IFN-γ and IL-4 in immunized sheep. Moreover, the VjbR antigen allowed serological differentiation between infected and vaccinated animals. These results suggest that M5-90Δ vjbR is an ideal live attenuated and efficacious live vaccine candidate against B. melitensis 16 M infection. Highlights • The M5-90Δ vjbR has low virulence in macrophages and mice. • The M5-90Δ vjbR can elicit an anti- Brucella -specific IgG response. • M5-90Δ vjbR imunizations induce a cellular response. • M5-90Δ vjbR imunizations induces protection against B. melitensis challenge. • M5-90Δ vjbR could be a potential DIVA vaccine. [ABSTRACT FROM AUTHOR]