The treatment against visceral leishmaniasis (VL) presents problems, mainly related to the toxicity and/or high cost of the drugs. In this context, a prophylactic vaccination is urgently required. In the present study, a Leishmania protein called LiHyE, which was suggested recently as an antigenic marker for canine and human VL, was evaluated regarding its immunogenicity and protective efficacy in BALB/c mice against Leishmania infantum infection. In addition, the protein was used to stimulate peripheral blood mononuclear cells (PBMCs) from VL patients before and after treatment, as well as from healthy subjects. Vaccination results showed that the recombinant (rLiHyE) protein associated with liposome or saponin induced effective protection in the mice, since significant reductions in the parasite load in spleen, liver, draining lymph nodes, and bone marrow were found. The parasitological protection was associated with Th1-type cell response, since high IFN-γ, IL-12, and GM-CSF levels, in addition to low IL-4 and IL-10 production, were found. Liposome induced a better parasitological and immunological protection than did saponin. Experiments using PBMCs showed rLiHyE-stimulated lymphoproliferation in treated patients’ and healthy subjects’ cells, as well as high IFN-γ levels in the cell supernatant. In conclusion, rLiHyE could be considered for future studies as a vaccine candidate against VL.