Protozoan parasites, such as Leishmania major (L. major), remained as a global health problem of the current century. Leishmania major is a major cause of cutaneous leishmaniasis (CL) in developed and developing countries. Traditionally, amphotericin B is prescribed as an alternative drug, while first-line drugs failed. Some active proteins of the innate immune system such as cathelicidins try to inhibit infection Via several proposed mechanisms. Here this research aimed to not only determine the antileishmanial activity of nano amphotericin B but also to evaluate which anti-leishmanial drug can induce the cathelicidin gene expression. Both promastigote and amastigote stages of L. major were exposed to various concentrations of nano amphotericin B, amphotericin B and finally compared to glucan time as standard drug for CL treatment. For the gene expression of cathelicidin, macrophages were exposed to the same concentration of anti-leishmanial drugs. The findings demonstrated that nano amphotericin B was more effective at all concentrations than amphotericin B. Additionally, among tested antileishmanial drugs, nano amphotericin B has more potency to induce the cathelicidin gene expression in macrophages cells. The findings revealed that nano amphotericin B has potential as an effective anti-leishmanial drug against CL caused by L. major parasites.