Growing evidence from both animal models and human intervention studies indicates that polyphenols can potentially attenuate postprandial glycemic responses and improve insulin sensitivity. A possible mechanism of action is through the inhibition of α‐glucosidases, critical for carbohydrate digestion in the gut, and by attenuation of glucose and fructose transport across small intestinal enterocytes. Sugar intake has increased over the last few decades, in part due to “hidden sugars” in foods, and a recent study demonstrated that excess sugar availability is a significant determinant of diabetes risk [1]. The research here focuses on the gut brush border enzyme sucrase which hydrolyses sucrose into glucose and fructose prior to absorption. We hypothesized that decreased expression or activity of sucrase would reduce the glycemic response to sucrose, potentially leading to positive effects on the glycemic response. The objective of the research is to evaluate if an extract from olive leaf, rich in the polyphenol oleuropein, chronically affects sucrose hydrolysis and transport.Fully differentiated Caco‐2/TC7 cells were used as a model for the small intestine and were exposed chronically to 1.5 mg/mL olive leaf extract (1.1 mM oleuropein) for the final 72 h of differentiation. This concentration is theoretically achievable in the gut lumen after consumption of the supplement “Bonolive”. An in vitro assay was developed using the cells as a source of sucrase to determine effects on activity and kinetic parameters. After treatments, sucrase mRNA was determined in whole cell lysates using droplet digital PCR, apical sucrase protein was quantitated after cell surface biotinylation using automated quantitative capillary Protein Simple Western blotting. Transport experiments were performed using cells cultured on porous supports, and the apical and basolateral concentrations of glucose and fructose were quantified using high performance anion exchange chromatography with pulsed amperometric detection.Olive leaf extract treatment reduced the Vmax of sucrase by 34%, decreased the specific activity by 32% and reduced total sucrase mRNA by 75% (all p<0.001). There was no change in total sucrase protein, but the proportion at the cell surface was lowered by 41% (p<0.001). Chronic treatment also affected sucrose hydrolysis and transport of the products; both apical glucose and fructose were attenuated by 37% and 21% respectively, in agreement with the data on sucrase activity. No change in glucose transport was observed when glucose was used as the substrate.In conclusion, chronic treatment by oleuropein‐rich olive leaf extract reduced the hydrolysis of sucrose. These results warrant future investigation into the use of olive leaf extract in humans for glycemic control after sugar consumption.Support or Funding InformationEuropean Research Council Advanced Grant “POLYTRUE?” (grant agreement 322467) and European Union Seventh Framework Programme (FP7/2007‐2013) “BACCHUS” (grant agreement 312090). Bonolive was kindly supplied by BioActor BV, The Netherlands. The Caco2/TC7 cell line was a kind gift by Prof. Monique Rousset, Centre de Recherche des Cordeliers, Paris, France.