Anthocyanins have multiple biological activities of benefit to human health. While a few studies have been conducted to evaluate the bioavailability of anthocyanins, the mechanisms of their absorption mechanism remain ill-defined. In the present study, we investigated the absorption mechanism of cyanidin-3-O-β-glucoside (Cy-3-G) in human intestinal epithelial (Caco-2) cells. Cy-3-G transport was assessed by measuring the absorptive and efflux direction. Inhibition studies were conducted using the pharmacological agents, phloridzin, an inhibitor of sodium-dependent glucose transporter 1 (SGLT1), or phloretin, an inhibitor of glucose transporter 2 (GLUT2). The results showed that phloridzin and phloretin significantly inhibited the absorption of Cy-3-G. In addition, Caco-2 cells transfected with small interfering RNA (siRNA) specific for SGLT1 or GLUT2 showed significantly decreased Cy-3-G absorption. These siRNA transfected cells also showed a significantly decreased rate of transport of Cy-3-G compared with the control group. These findings suggest that Cy-3-G absorption is dependent on the activities of SGLT1 and GLUT2 in the small intestine and that SGLT1 and GLUT2 could be a limiting step for the bioavailability of Cy-3-G.