Introduction: TOTUM-070 is a patented polyphenol-rich compound which has shown hypocholesterolemic properties in preclinical studies. However, clinically validated approaches and further investigations on the mode of action at cellular level especially in humans are required for optimized care management. In this study, we designed an ex-vivo clinical innovative approach considering the metabolites produced by the digestive tract following the ingestion of TOTUM-070 in humans.Methods: Human serum was collected in healthy subjects before and following acute intake of 5g of TOTUM-070. Availability of circulating metabolites was confirmed and characterized by UPLC-MS. Human serum enriched with metabolites deriving from TOTUM-070 absorption was further incubated with human hepatocytes, pretreated or not with palmitate (250 μM). In such lipotoxic environment, hepatocyte behavior was monitored to determine whether and how TOTUM-070 metabolites may improve cholesterol metabolism in human.Results: In the presence of the human metabolites from TOTUM-070, human hepatocytes were protected from an induced lipotoxic stress. No effect on cell toxicity was detected in the presence of enriched sera. Hepatocyte protection was characterized by (1) the inhibition of both triglycerides (-41%, p<0.001) and cholesterol (-50%, p<0.001) storage, (2) a reduced de novo cholesterol synthesis (HMG-CoA reductase activity reduced by 44%, p<0.001), (3) a lowered fatty acid synthase expression (p<0.001), (4) the stimulation of the Lecithin Cholesterol Acyl Transferase (LCAT) activity (p<0.05) and finally (5) a higher CYP7A1 expression (p<0.05). All together, these data provide new biochemical insights into the mechanisms underlying, in humans, the beneficial impact of TOTUM-070 on lipid metabolism in liver cells.Conclusion: Using a pioneering clinical ex vivo approach considering the digestive processes of nutrients, we give clues on the role of circulating metabolites produced following TOTUM-070 intake in humans in hepatocyte protection.