Life-long immunity to leishmaniasis in recovered patients has inspired the development of vaccines against disease. The present study aimed to evaluate a non-described hypothetical Leishmania amastigote-specific protein, identified by an immunoproteomic approach in L. infantum, attempting to select a new candidate antigen for specific serodiagnosis and a vaccine against visceral leishmaniasis (VL). The recombinant protein (rLiHyp1) was recognized by antibodies from sera of asymptomatic and symptomatic canine visceral leishmaniasis (CVL), but presented no cross-reactivity with sera of vaccinated dogs, those with Chagas' disease or healthy animals. In addition, the rLiHyp1 plus saponin was able to induce a Th1 response, which was based on the production of high levels of IFN-γ, IL-12, and GM-CSF after in vitro stimulation in BALB/c mice. The protective efficacy of rLiHyp1 plus saponin was evaluated in mice challenged with L. infantum promastigotes. Challenged and vaccinated mice showed significant reductions in the number of parasites in all evaluated organs, and the protection was associated with a Th1-type response. Therefore, the present study reveals a new potential candidate for the improvement of serodiagnosis of CVL, as well as an effective vaccine candidate against VL.