As an effort towards vaccine development against African trypanosomiasis, we studied key parasite molecules that mediate VSG functions, specifically the major surface protease-B of Trypanosoma brucei that catalyzes proteolytic removal of old VSGs for expression of new ones, an important stage-specific function that allows the parasite to survive in its host, thus making it an attractive candidate for vaccine development. Herein, Tbmsp-b gene was cloned into a pVAX-1 plasmid to produce pVAX-1-Tbmsp-b construct for DNA vaccine trials. BALB/c mice were immunized by intradermal injection with 100 µg dose of the construct thrice on days 0, 21 and 42, then inoculated with 2000 parasites on day 56. Anti-trypanosomes specific antibody (IgG) and cytokine (γ-IFN) were monitored by ELISA from sera of immunized and unimmunized mice. Immunized mice showed significantly (p < 0.05) higher IgG and γ-IFN responses, lower parasitaemia (by 75% and 51.2% of parasitaemic scores on first and fifth week of infection) and longevity by up to 22 days compared to unimmunized mice. These results showed that the construct provided partial protection to virulent T. b. brucei (Federe strain) infection in susceptible BALB/c mice suggesting the potentials for using MSP-B as an antigen in DNA vaccine development against African trypanosomiasis.