• Preussin derivatives, asperidine B and its desmethyl analogue, exerted antioxidative and lipid-lowering effects similarly to their parent compound. • Asperidine B had higher potency by lowering cholesterol level than its methyl analogue in both ex vivo isolated jejunal epithelium, and in vivo models. • Asperidine B upregulated α-catenin and EpCAM proteins by proteomics approach, which they are relevance in tight junction integrity. • Asperidine B strongly inhibited intestinal cholesterol absorption by directly interaction with ZO-1 protein function and expression.