Background: Leishmaniasis is a vector-borne disease prevalent in 98 countries worldwide. The current treatment has shortcomings, including drug resistance and adverse effects, highlighting the need for novel medications and treatment strategies. This study aimed to investigate the anti-leishmanial effect of thymoquinone (TQ) during the regulation of autophagy in the macrophage cell line (RAW 264.7). Methods: After culturing the macrophage cell line, an MTT assay was performed to assess the cytotoxicity effects of the agents at different concentrations of TQ, HCQ (hydroxychloroquine), MET (metformin), and GLU (glucantime). The study groups included PBS, GLU, TQ, TQ + MET, GLU + MET, TQ + HCQ, GLU + HCQ, HCQ, and MET. The cells were then infected with L. major and treated with TQ, while autophagy was regulated using HCQ and MET. Subsequently, the infection index, the number of amastigote loads, and the fold change in the expression of specific autophagy-related genes (LC3, P62, and Beclin) in the treatment groups were evaluated. Results: There was a significant decrease in the percentage of the infected macrophages treated with TQ and also the autophagy inhibitor HCQ compared to the control group. Macrophages treated with HCQ + TQ showed a significant reduction in the infection index and amastigote load compared to the TQ-treated group. Additionally, using HCQ as an autophagy inhibitor, along with TQ or GLU, enhanced the clearance of parasites and reduced the infection index of macrophages. Conclusion: Downregulating autophagy could be a promising approach for Leishmania therapy, by which the leishmanicidal effect of TQ and GLU will be enhanced.

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