Abstract In recent years, research has focused on the immunoreactive components of the S. schenckii cell wall that can be relevant targets for preventive and therapeutic vaccines against sporotrichosis, an emergent worldwide mycosis. In previous studies, we identified a 47-kDa enolase as an immunodominant antigen in mice vaccinated with purified fungal wall proteins and adjuvants. In this study, the immunolocalization of this immunogen in the cell wall of S. schenckii and S. brasiliensis is shown for the first time. In addition, a recombinant enolase of Sporothrix spp (rSsEno) was studied with the adjuvant Montanide Pet-GelA (PGA) as a vaccine candidate. The rSsEno was produced with high purity. In addition, mice immunized with rSsEno plus PGA showed increased antibody titers against enolase and increased median survival time comparedto nonimmunized or rSsEno-immunized mice. Enolase immunization induced a predominant T-helper-1 (Th1) cytokine pattern in splenic cells after in vitro stimulation with rSsEno. Elevated production of interferon-γ (IFN-γ) and interleukin-2 (IL-2) was observed with other cytokines involved in the innate immune defense, such as TNF-alpha, IL-6, and IL-4, which are necessary for antibody production. These results suggest that we should continue testing this antigen as a potential vaccine candidate against sporotrichosis.