Brucellosis is one of the most common zoonotic diseases worldwide, caused by the infection of Brucella spp. Despite of extensive research, till now no licensed vaccines are available against Brucella infection for human application. In the present study, a recombinant chimeric protein rGB contriving immunologically active regions of Gapdh and bvrS from B. abortus S19 was constructed, cloned and expressed in E. coli host cell. Group of mice immunized with purified rGB protein elicited both Th1 and Th2 mediated immunity as indicated by the higher secretion of IgG2a and IgG1 antibody isotypes in the sera. This observation was further supported by the elevated cytokine profiles of IFN-γ and IL-2 reflecting Th1 and IL- 4 as an indicator of Th2 response upon stimulation of splenocytes with purified rGB antigen. The in-vitro lymphocyte proliferation assay resulted in a higher splenic lymphocyte response in immunized mice upon antigen stimulation in comparison with control mice implicating the development of elevated immune responses. Macrophage monolayer supplemented with anti-rGB polysera illustrated efficient protection (>85% survival, P < 0.001) against challenge of B. abortus strain 544. These findings demonstrate the potential efficacy of fusion protein rGB as a candidate subunit vaccine and showed the development of memory response and functional role of antibodies in protection against Brucella infections.