The global prevalence of HIV is a major challenge for the control of visceral leishmaniasis. Although the effectiveness and usefulness of amprenavir (APV) are well studied in anti-retroviral regimens, very little is known on HIV/VL-co-infected patients. In the present study, we report for the first time the protective efficacy of APV against visceral leishmaniasis by inhibition of DNA Topoisomerase I (LdTOP1LS) and APV-induced downstream pathway in programmed cell death (PCD). During the early phase of activation, reactive oxygen species (ROS) is increased inside the cells, which causes subsequent elevation of lipid peroxidation. Endogenous ROS formation and lipid peroxidation cause eventual depolarization of mitochondrial membrane potential (ΔΨ). Furthermore, the release of cytochrome c and activation of CED3/CPP32 group of proteases lead to the formation of oxidative DNA lesions followed by DNA fragmentation. The promising in vitro and ex vivo results promoted to substantiate further by in vivo animal experiment, which showed a significant reduction of splenic and hepatic parasites burden compared to infected controls. Interestingly, APV selectively targets LdTOPILS and does not inhibit the catalytic activity of human topoisomerase I (hTopI). Moreover, based on the cytotoxicity test APV is not toxic for host macrophage cells, which is correlated with non-responsiveness of inhibition of catalytic activity of hTopI. Taken together, this study provides the opportunity for discovering and evaluating newer potential molecular therapeutic targets for drug designing. The present study might be exploited in future as important therapeutics, which will be useful for treatment of VL as well as HIV-VL co-infection.