The leishmaniases are a complex of vector-borne diseases caused by protozoan parasites of the genus Leishmania. LEISHDNAVAX is a multi-antigen, T cell epitope enriched DNA vaccine candidate against human leishmaniasis. The vaccine candidate has been proven immunogenic and showed prophylactic efficacy in preclinical studies. Here, we describe the safety testing of LEISHDNAVAX in naïve mice and rats, complemented by demonstration of tolerability in Leishmania infected mice. Biodistribution and persistence were examined following single and repeated intradermal administration to rats. DNA vectors were distributed systemically but did not accumulate upon repeated injections. While vector DNA was cleared from most other tissues within 60 days after the last injection, it persisted in skin at the site of injection and in draining lymph nodes. Evaluation of single-dose and repeated-dose toxicity of the vaccine candidate after intradermal administration to naïve, non-infected mice did not reveal any safety concerns. LEISHDNAVAX was also well tolerated in Leishmania infected mice. Taken together, our results substantiate a favorable safety profile of LEISHDNAVAX in both naïve and infected animals and thus, support the initiation of clinical trials for both preventive and therapeutic applications of the vaccine.Gene Therapy accepted article preview online, 14 April 2015. doi:10.1038/gt.2015.35.