Previous in vivo studies including those with knockout mice suggested that Niemann-Pick C1-like 1 (NPC1L1) plays an essential role in the intestinal absorption of cholesterol. To characterize the mechanism of cholesterol uptake mediated by NPC1L1, an in vitro system reflecting the function of this transporter needs to be established. In the present study, we constructed NPC1L1 overexpressing CaCo-2 cells as an in vitro model and characterized the transport properties of NPC1L1. Immunohistochemical staining revealed that CaCo-2 cells express NPC1L1 on the apical membrane. It was also demonstrated that the uptakes of both cholesterol and beta-sitosterol are increased by NPC1L1 overexpression. In addition, the uptake of cholesterol was increased in a dose-dependent manner by an increase in the content of taurocholate in micelles, whereas micellar phosphatidylcholine showed a negative correlation with cholesterol uptake. Furthermore, it was confirmed that sterol uptake increased by NPC1L1 overexpression was inhibited by ezetimibe. We could thus establish an in vitro intestinal model to study the mechanism of NPC1L1-dependent sterol uptake and to screen drug candidates whose target is NPC1L1.