Caco-2 cell model for the transport of glucoside compounds was established to provide the basis for studying intestinal absorption of glucose and glucoside compounds. A reliable cell model was established by selecting the best medium for Caco-2 cells, measuring the single layer density, detecting monolayer permeability and the activity ratio of alkaline phosphatase on both sides of the cell, observing the single layer sub microstructure, and analyzing the mRNA and protein expression levels of SGLT1 and GLUT2. The results showed that the optimal medium for Caco-2 cells was DMEM medium containing low glucose and supplying 15% serum. The trans epithelial electrical resistance of Caco-2 cells tended to be stable in 14-17 days, and the apparent permeability coefficient of phenol red reached 0.49×10-6-0.51×10-6cm·s-1. The microvilli differentiated and matured, and the ratio of alkaline phosphatase on both sides of cells reached more than 2.0. The mRNA expression of glucose transporter SGLT1 was significantly higher on 14d than those on 17d and 21d, while the protein expression of glucose transporter SGLT1 and GLUT2 had no significant difference on 14d, 17d and 21d (P<0.05). Caco-2 cells could be cultured for 14-17 days to establish a reliable intestinal absorption model, which might be used to study the absorption of glucose and glucoside compounds in vitro.