Cell-mediated responses while females develop stronger humoral responses suggesting that males are more resistant to infection. However, conflicting reports also exist as in case of visceral leishmaniasis whether caused by Leishmania donovani in humans or Leishmania infantum in dogs susceptibility is higher in males than females. Similarly, in case of L. donovani infection in hamster model, male developed more parasites than females which decreased by castration while ovarectomy in females promoted infection. Similar differences also exist in different mouse strains, while male Balb/c and DBA/2 mice are more susceptible to systemic infection with Leishmania major following intravenous inoculation as compared to females; male C57Bl/ 10 and DBA/2 mice are more resistant than females to subcutaneous challenge with this parasite. Undoubtedly, the parasite species initiating infection, the tissue site involved and the host species are amongst the variable factors influencing these observed differences. In light of these conflicting reports, we included both sexes of mice and hamsters in our study and only those animals were included that had grade I infection. Our results demonstrated that inhibition of parasite burden during combination therapy with lipo-CpG-ODN-2006 plus sub-curative dose of miltefosine in both rodent models is associated with induction of strong cell-mediated immune responses including Th1 cytokine synthesis, NO generation, and robust lymphocyte proliferation along with induction of Leishmania- specific antibody responses. Several reasons limit the use of miltefosine monotherapy. Some of these include teratogenic effect in pregnant women, fear of resistance and its long half-life in humans. In contrast, combination therapy is advantageous over monotherapy as it delays or prevents the emergence of resistance and requires lower and shorter dose regimen against various infectious diseases. In the present study, we explored the synergy between chemotherapy and host immune function by using CpG-ODN-2006 in combination with miltefosine. Interestingly, assessment of splenic infection at later time point in infected hamsters, that underwent combination therapy, revealed almost complete absence of parasite burden in spleen cells, which further suggested that this therapy is also effective in chronic model of experimental VL. On a contrary, a moderate increase in parasite burden in hamsters treated with free and liposomal forms of CpG-ODN-2006 was observed. The increase in parasite burden was also examined in hamsters treated with sub-curative and curative doses of miltefosine, which showed 14% and 2% increase, respectively, on day 30 post treatment. Similar trend were also observed at day 30 post treatment in L. donovani infected Balb/c mice that underwent combination therapy. This piece of evidence also suggests that lipo-CpG-ODN-2006 with subcurative miltefosine boosts host immunity which provides long term protection in chronic model of experimental VL.