Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa transmitted by infected sand flies. Today this disease is spread in more than 88 countries and affecting more than 320 million people. There is no licensed vaccine available against human leishmaniasis. We have previously established a CRISPR genome-edited attenuated Leishmania major strain (LmCen-/-) as a second-generation leishmanization vaccine candidate. We evaluated the efficacy of LmCen-/- against VL caused by L.donovani infection in a pre-clinical hamster model as well as immunogenicity in the PBMCs isolated from human subjects living in an endemic region. Intradermal immunization of hamsters with LmCen-/- did not develop any lesion; while inducing a strong pro-inflammatory immune response. Immunized hamsters when challenged with L. donovani either by intradermal needle injection or by infected sand flies were protected as indicated by a lack of pathology and mortality. Spleen cells from LmCen-/- immunized and challenged hamsters produced significantly higher Th1-associated cytokines including IFN-γ and TNF-α, and significantly reduced expression of the anti-inflammatory cytokines IL-10 and IL-21, compared to non-immunized and challenged animals. We developed a GLP-grade LmCen-/- which is equally protective as laboratory-grade LmCen-/- parasites. GLP-grade LmCen-/- parasites induced a pro-inflammatory immune response in the PBMCs isolated from healed VL patients as well as healthy people living in the endemic region. Together, this study demonstrates that the LmCen-/- parasites are safe and efficacious against VL and can proceed to be tested as a vaccine in human clinical trials. These findings could help in the development and evaluation of effective Leishmania vaccines against Leishmaniasis.