The dark blue pigment (DBP) is a health ingredient from Vaccinium bracteatum Thunb. leaves used as a functional food supplement. However, the details of transepithelial absorption on the intestinal epithelial cells are barely understood. This study aimed to clarify the absorption properties of DBP in the Caco-2 cell monolayer model and evaluate the effect on the endo-metabolism and barrier function of Caco-2 cells. The results showed that the DBP did not show the dose-dependent toxic effect to Caco-2 cells between 0.25 and 1.5 mg/mL, which did not cause disorder in the normal cell metabolism and absorption activity. The Caco-2 cells monolayer model could absorb DBP by passive and active transport, and the absorptive pattern was dose-dependent when the concentration was more than 0.25 mg/mL. During DBP absorption, an increase in mRNA and protein expressions of glucose transporters demonstrated that the glucose transporters were the potential transporter of DBP. But the glucose transport amounts were significantly lowered after 30 min of DBP treatment, indicating that DBP owned the inhibitory effect on glucose transportation. Furthermore, DBP also owned protective effects on the barrier function of intestinal epithelial cells.