Miltefosine is the new drug of choice for the treatment of leishmaniasis. The aim of this work was to study the molecular mechanisms and immunomudolatory properties of miltefosine in J774 cell line infected with the Leishmania major (MRHO/IR/75/ER) parasite. In this experimental study infected J774 cell line by L. major, treated by miltefosine and incubated for 72 h. Total RNA was extracted and cDNA was synthesis. RT- PCR was used for study of IFN? and iNOS gene expression. IFN? and iNOS proteins were analyzed by Western blotting. Cell culture supernatant was examined by ELISA for of IL12 and IL10 concentration. After 4 h incubation, miltefosine increased iNOS and IFN? gene expression in L. major infected J774 cell line. Western blot analysis of extracted cell proteins showed 130 and 17 kDa of bonds related to iNOS and IFN?, respectively. After 48 h treatment with miltefosine, analysis of cell supernatant with ELISA showed a significant increase of IL12 but no change in IL10 Cytokine. Study showed that miltefosine in addition to its direct effect can improve cellular immunity with rising of IFN? and of iNOS genes expression that are able to activate macrophages.