ObjectiveThis study aimed to investigate the antioxidant, antihyperlipidemic, antihypercholesterolemic, and hepatoprotective properties of Acacia nilotica (A. nilotica) pod extracts in male albino Wistar rats.MethodsA. nilotica pod extracts (ANPE) were prepared using ethanol, methanol, acetone, and distilled water. Antioxidant properties were evaluated by determining total phenolic content (TPC), ferric reducing antioxidant power (FRAP) and DPPH (2,2-diphenyl-1-picrylhydrazyl) activity. ANPE were administered at doses of 250, 500, and 750 mg/kg/day to rats with hypercholesterolemia induced by a high-fat diet. Lipid profile, cardiac enzymes (CK-MB and Troponin-I) and oxidative stress markers were measured. In addition, virtual screening and molecular dynamic simulations were performed to assess the potential of A. nilotica pod constituents to inhibit oxidosqualene cyclase (OSC), a key enzyme in cholesterol biosynthesis.ResultsThe ethanolic extract of A. nilotica pods displayed the highest antioxidant activity with a TPC of 61.83 ± 2.60 mg GAE/g, FRAP of 1712.9 ± 4.11 µg Fe/g and DPPH inhibition of 79.47% ± 0.66%. The highest dose (750 mg/kg) of ANPE significantly decreased serum cholesterol and LDL levels, while improving HDL and HDL/LDL ratio (p < 0.05). This dose also improved oxidative stress markers, cardiac enzymes, the atherogenic index, and the Castelli index compared with the hypercholesterolemic control group (p < 0.05). Histopathological examination of liver and kidney tissues confirmed that the highest dose of APNE exerted marked hepatoprotective and renoprotective effects (p < 0.05). In silico analyses highlighted a plausible molecular mechanism whereby β-sitosterol of A. nilotica interacts with the OSC active site, supporting a hypothesis for the observed reduction in cholesterol biosynthesis.ConclusionANPE exhibits strong antihypercholesterolemic, antioxidant, hepatoprotective, and nephroprotective effects in a rat model of diet-induced hypercholesterolemia. The long-term safety and efficacy of its bioactive constituents should be evaluated in future preclinical and clinical studies. Computational modelling suggests that β-sitosterol from A. nilotica may act as a putative OSC inhibitor, providing a mechanistic framework that warrants further in vitro and biochemical validation.