Paratuberculosis, also called as Johne's Disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis(MAP), and development of improved vaccines is urgently required considering its economic importance. Here, we report the cloning and expression of a 74kDa recombinant fusion protein (Map 74F) and evaluation of its protective efficacy against MAP challenge in mice. Map74F was generated by the sequential linkage in tandem of the ORFs of the~17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C terminus with ~14.6-kDa N-terminal portion of Map3527. C57BL/6 mice immunized with Map74F along with MPL+TDM emulsion had a significant IgG1 response but not IgG2a. In vaccinated animals, the IgG1/IgG2_a ratio which increased until 4 wk after MAP challenge, decreased gradually from 8 wk indicating a possible shift to a Th1 response. Antigen specific IFN-γ responses measured by IFN-γ ELISA and ELISPOT assay were significantly higher in mice immunized with Map 74F than the control animals. The results revealed that IL-4 and IL-10 mRNA expression by spleen cells from immunized animals were higher than that of control animals, whereas no significant differences were detected in the expression levels of other cytokine genes including IL-2, IL-12, TNF-αand IFN-γ . Antigen specific CD3⁺ and CD4⁺ T cell populations increased significantly in mice immunized with Map 74F, whereas, no significant differences were detected in the CD8⁺ T cell populations between the immunized and control animals. Following challenge, MAP load was significantly lower in spleen, liver and mesenteric lymph nodes of immunized animals compared to the control animals indicating protection against MAP infection. This was further evident by the improved spleen and liver pathology of the immunized animals which had fewer granulomas and lesser numbers of acid-fast bacilli. Results of our study indicated that immunization of mice with Map74F protected mice against MAP infection.