<jats:title>Abstract</jats:title><jats:p>Leishmaniasis, a major neglected tropical disease, affects millions of individuals worldwide. Among the various clinical forms, visceral leishmaniasis (VL) is the deadliest. Current antileishmanial drugs exhibit toxicity- and resistance-related issues. Therefore, advanced chemotherapeutic alternatives are in demand, and currently, plant sources are considered preferable choices. Our previous report has shown that the chloroform extract of <jats:italic>Corchorus capsularis</jats:italic> L. leaves exhibits a significant effect against <jats:italic>Leishmania donovani</jats:italic> promastigotes. In the current study, bioassay-guided fractionation results for <jats:italic>Corchorus capsularis</jats:italic> L. leaf-derived β-sitosterol (β-sitosterol<jats:sub>CCL</jats:sub>) were observed by spectroscopic analysis (FTIR, <jats:sup>1</jats:sup>H NMR, <jats:sup>13</jats:sup>C NMR and GC–MS). The inhibitory efficacy of this β-sitosterol<jats:sub>CCL</jats:sub> against <jats:italic>L. donovani</jats:italic> promastigotes was measured (IC<jats:sub>50</jats:sub> = 17.7 ± 0.43 µg/ml). β-Sitosterol<jats:sub>CCL</jats:sub> significantly disrupts the redox balance via intracellular ROS production, which triggers various apoptotic events, such as structural alteration, increased storage of lipid bodies, mitochondrial membrane depolarization, externalization of phosphatidylserine and non-protein thiol depletion, in promastigotes. Additionally, the antileishmanial activity of β-sitosterol<jats:sub>CCL</jats:sub> was validated by enzyme inhibition and an in silico study in which β-sitosterol<jats:sub>CCL</jats:sub> was found to inhibit <jats:italic>Leishmania donovani</jats:italic> trypanothione reductase (<jats:italic>Ld</jats:italic>TryR). Overall, β-sitosterol<jats:sub>CCL</jats:sub> appears to be a novel inhibitor of <jats:italic>Ld</jats:italic>TryR and might represent a successful approach for treatment of VL in the future.</jats:p>