Leishmaniasis is a vector-borne disease in tropical and subtropical regions. It presents a wide spectrum of clinical manifestations; Cutaneous Leishmaniasis (CL) is the most common form leading to a skin nodule. L-Arginine (L-Arg) is an important amino acid involved in many metabolic pathways in host macrophages (MΦs) including NO synthesis. The L-Arg pathway is relevant to leishmaniasis due to its role in regulating MΦ functions. Activated MΦs can produce leishmaniacidal molecules, such as Nitric Oxide (NO) and oxidative mediators to kill parasite. Different concentrations of L-Arg were used; their toxicities assessed in naïve Balb/c mice. The highest concentration with lowest toxicity was selected to apply in Leishmania major infected mice. Test group was injected with three types of L-Arg (oral, local, injection) and control group received normal saline. Then, the lesion size, the measurement of amastigotes proliferation in MΦ, the pathophysiology of mice (hepato/spelenomegaly, survival rate, body weight) was all evaluated. In addition, plasma and tissue suspensions were investigated for NO induction using Griess Micro Assay (GMA). This is the first application of oral, local and injection forms of L-Arg against Iranian strain L. major MRHO/IR/75/ER. Results indicated L-Arg had ability to elevate NO in murine host. No pathological effects were observed in oral, local and injection types of L-Arg. Moreover, a significant decrease in parasite proliferation was observed only in oral group which presented anti-leishmanial activity by reduction liver and spleen positive smears. In injection form, percentage of positive smears was reduced only in spleen; a reduction observed in lesion sizes after treatment with oral and injection form of L-Arg; no significant alteration of local L-Arg to limit lesion size in CL was indicated here. The L-Arg is NO precursor and has been used safely for decades to treat physiological condition and used as food supplementary with no toxicity. In this study, L-Arg presented its partial ability to induce NO and treat animals. It also has limited potential therapeutic effects against CL by alteration of NO in host; therefore, it may be indicated solo as an anti-leishmanial agent or in a combination therapy for CL in mice. This may be the first immunotherapy trial against CL in Iran.