Background: Canine visceral leishmaniasis (CVL) caused by Leishmania infantum is endemic in the northwest and south of Iran. An appropriate vaccine can help to prevent and control visceral leishmaniasis in both humans and animals. Few studies have confirmed that the fucose-mannose ligand (FML) antigen of Leishmania donovani produced protective immunity in dogs against CVL. Objective: To evaluate the immune responses of vaccinated dogs against FML antigen of L. infantum. Methods: We isolated the FML antigen from native L. infantum and vaccinated the dogs with FML-saponin in an endemic area of VL in Iran to evaluate the immune responses of vaccinated dogs against this antigen. Results: Our results indicated a significant increase in the expression of IFN-γ, IL-10 and IL-13, but not IL-12A, gene transcripts in PBMCs of FML-saponin vaccinated dogs in comparison with controls. Our findings showed a significant difference in the ratio of IFN-γ/IL-10 mRNA expression in FML-saponin vaccinated dogs in comparison with two control groups. Moreover, a significant level of anti-FML antibodies was detected in serum of vaccinated dogs. Conclusion: These findings showed that FML-saponin stimulates both Th1 and Th2 immune responses with predominant Th1 and strong humoral immune responses to produce protective immunity against CVL.