Brucellosis, which is caused by Brucella spp., is a zoonotic infectious disease that can cause great hazard to public health and safety. The virulence of Brucella is essential for survive and multiply in host macrophages. GntR is a transcriptional regulator in Brucella that is required for virulence in macrophages and mice, and involved in resistance to stress responses. To determine the expression levels of target genes of GntR, we detected the expression levels of the GntR target genes in Brucella infected BALB/c mice. The results showed that several genes related to virulence, including omp25, virB1, vjbR, dnaK, htrA and hfq, were regulated by GntR during infection in BALB/c mice. Moreover, the 2308ΔgntR mutant induced high protective immunity in BALB/c mice challenge with B. abortus 2308 (S2308), and elicited an anti-Brucella-specific immunoglobulin G (IgG) response and induced the secretion of gamma interferon (IFN-γ) and interleukin-4 (IL-4). All together, these results indicated that gntR promoted the virulence of Brucella. The 2308ΔgntR was significantly attenuated in macrophages and mice and induced protective immune response during infection, suggested that 2308ΔgntR mutant is an attractive candidate for the design of a live attenuated vaccine against Brucella.