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Cardiovascular disease risk is related to oxidative stress and hypercholesterolemia. Guarana seed powder contains flavanols that possess antioxidant properties and cholesterol-lowering effects. However, the molecular mechanism through which guarana seed powder may decrease cholesterol uptake from the intestinal lumen remains unclear. Thus, this study aimed to investigate the effect of guarana powder aqueous extract on cholesterol absorption mechanisms. After simulated gastrointestinal digestion, we performed assays to determine enzymatic inhibitory capacity, bile acid binding capacity, and cholesterol micellar solubilization. Caco-2 cells were used for permeation and protein identification assays. Digested guarana powder extract inhibited pancreatic lipase in a dose-dependent manner (half-maximal inhibitory capacity: 1.033 µg/mL) and, at a concentration of 1 mg/mL, bound 45.63 % of sodium taurocholate and decreased cholesterol micellar solubilization by 10.14 %. Moreover, incubation with the extract reduced cholesterol absorption by Caco-2 cells and decreased intracellular cholesterol transporter levels. These results indicate that guarana seed powder have potential applications for blood cholesterol management, presenting hypocholesterolemic effects owing to the presence of bioacessible polyphenols.