Leishmaniasis is a group of neglected tropical diseases (NTDs) caused by about 20 species of obligate intracellular protozoan parasites of the genus Leishmania, which occurs in cutaneous, mucocutaneous, and visceral forms. Many researchers have sought to utilize natural products for novel and effective treatments to combat many infectious diseases, including leishmaniasis. Holarrhena pubescens Wall. ex G. Don (Apocynaceae) bark is a rich source of bioactive steroidal alkaloids. The total alkaloidal extract (IC50 6.12 +/- 0.117 mu g/mL), and the isolated alkaloid, holanamine, showed significant antileishmanial activity (IC50 2.66 +/- 0.112 mu M against AG83 and 3.80 +/- 0.126 mu M against BHU-575) against the Leishmania donovani parasite, better than miltefosine (IC50 19.61 +/- 0.093 mu M against AG83 and 23.20 +/- 0.094 mu M against BHU-575). Holanamine inhibited the L. donovani topoisomerase 1B (LdToP1B) in a non-competitive manner (IC50 2.81 +/- 0.105 mu M), indicating that it interacts with the free enzyme and enzyme-DNA complex without inhibiting human topoisomerase. Hydrogen bonding and hydrophobic interactions of holanamine with the N-terminal and hinge region of the large subunit of LTop1B is responsible for its potent antileishmanial activity, as shown by docking studies. Treatment with holanamine causes apoptotic-like cell death by generating cellular and mitochondrial reactive oxygen species, disrupting the mitochondrial membrane potential and inducing ultrastructural alterations in the promastigotes. Holanamine effectively clears intracellular amastigotes but minimally affects host macrophages with no significant cytotoxicity in HEK 293 and L929 cell lines. Thus, our studies show that holanamine can further be used to develop effective antileishmanial agents against evolving drug-resistant parasites.