Brucella (B) species are brucellosis causative agents, a worldwide zoonotic illness causing Malta fever in humans and abortion in domestic animals. In this work, we evaluated the vaccine potential of Trimethyl chitosan (TMC) nanoparticles formulation of Urease (TMC/Urease) against brucellosis. TMC/Urease nanoparticles and urease without any adjuvant were separately administered both orally and intraperitoneally. Intraperitoneal (i.p.) administration of urease alone as well as oral administration of both TMC/Urease nanoparticles and urease alone, elicited low titers of specific immunoglobulin G (IgG), while i.p. immunization with TMC/Urease nanoparticles induced high specific IgG production levels. As it was indicated by the cytokine assay and the antibody isotypes, i.p. immunization by urease alone, and TMC/Urease nanoparticles induced a mixed Th1-Th2 immune response, whereas oral administration of both urease alone and TMC/Urease nanoparticles induced a mixed Th1-Th17 immune response. In lymphocyte proliferation assay, spleen cells from i.p.-vaccinated mice with TMC/Urease nanoparticles showed a strong recall proliferative response. Vaccinated animals were challenged with virulent strains of B. melitensis and B. abortus. I.p. vaccination with TMC/Urease nanoparticles resulted in a high degree of protection. Altogether, our results indicated that TMC nanoparticles are a potent delivery system for i.p.-administered Brucella antigens.