Although polyphenols inhibit glucose absorption and transport in vitro, it is uncertain whether this activity is sufficient to attenuate glycaemic response in vivo. We examined this using orange juice, which contains high levels of hesperidin. We first used a combination of in vitro assays to evaluate the potential effect of hesperidin and other orange juice components on intestinal sugar absorption and then tested whether this translated to an effect in healthy volunteers. Hesperidin attenuated transfer of 14C-labelled glucose across differentiated Caco-2/TC7 cell monolayers. The involvement of the sugar transporter GLUT2 was demonstrated by experiments carried out in the absence of Na to exclude the contribution of sodium-glucose linked transporter 1 and further explored by the use of Xenopus laevis oocytes expressing human GLUT2 or GLUT5. Fructose transport was also affected by hesperidin partly by inhibition of GLUT5, while hesperidin, even at high concentration, did not inhibit rat intestinal sucrase activity. We conducted three separate crossover interventions, each on ten healthy volunteers using orange juice with different amounts of added hesperidin and water. The biggest difference in postprandial blood glucose between orange juice and control, containing equivalent amounts of glucose, fructose, sucrose, citric acid and ascorbate, was when the juice was diluted (ΔCmax=–0·5 mm, P=0·0146). The effect was less pronounced when the juice was given at regular strength. Our data indicate that hesperidin can modulate postprandial glycaemic response of orange juice by partial inhibition of intestinal GLUT, but this depends on sugar and hesperidin concentrations.