Abstract Background Visceral leishmaniasis is the most severe form of leishmaniasis and no effective vaccine exists. The use of live attenuated vaccines is emerging as a promising vaccination strategy. Results In this study, we tested the ability of a Leishmania infantum deletion mutant, lacking both HSP70-II alleles (ΔHSP70-II), to provide protection against Leishmania infection in the L. major-BALB/c infection model. Administration of the mutant line by either intraperitoneal, intravenous or subcutaneous route invariably leads to the production of high levels of NO and the development in mice of type 1 immune responses, as determined by analysis of anti-Leishmania IgG subclasses. In addition, we have shown that ΔHSP70-II would be a safe live vaccine as immunodeficient SCID mice, and hamsters (Mesocricetus auratus), infected with mutant parasites did not develop any sign of pathology. Conclusions The results suggest that the ΔHSP70-II mutant is a promising and safe vaccine, but further studies in more appropriate animal models (hamsters and dogs) are needed to appraise whether this attenuate mutant would be useful as vaccine against visceral leishmaniasis. ; This work was supported by grants from the Ministerio de Ciencia y Tecnología (BFU2009-08986) and the Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD06/0021/0008-FEDER) to JMR, and Comunidad Autónoma de Madrid (S-SAL-0159-2006), Ministry of Science and Innovation of Spain (SAF2007-61716 and SAF2010-18733), “Red Tematica de Investigación en Enfermedades cardiovasculares” (RECAVA RD06/0014/1013); “Red de Investigación de Centros de Enfermedades Tropicales” (RICET RD06/ 0021/0016) to MF. Also, an institutional grant from Fundación Ramón Areces is acknowledged.