OBJECTIVE: To evaluate the efficacy and immunogenicity of the zeta domain over-expressing Leishmania donovani (Ld_ζ1 METHODS: In this study, Leishmania overexpressor Ld_ζ1 RESULTS: Ld_ζ1 CONCLUSION: Our findings demonstrated that overexpressing the zeta toxin resulted in controlled parasite attenuation, lowering pathogenicity while retaining immunogenic features. Our work established the zeta over-expressor's protective efficacy, immunogenicity, and proliferation in response to a Leishmania challenge in vitro and in vivo. This preliminary prototype study suggested that Ld_ζ1