In polarized hepatocytes, the predominant route for apical resident proteins to reach the apical bile canalicular membrane is transcytosis. Apical proteins are first sorted to the basolateral membrane from which they are internalized and transported to the opposite surface. We have noted previously that transmembrane proteins and GPI-anchored proteins reach the apical bile canaliculi at very different rates. Here, we investigated whether these differences may be explained by the use of distinct endocytic mechanisms. We show that endocytosis of both classes of proteins at the basolateral membrane of polarized hepatic cells is dynamin dependent. However, internalization of transmembrane proteins is clathrin mediated, whereas endocytosis of GPI-anchored proteins does not require clathrin. Further analysis of basolateral endocytosis of GPI-anchored proteins showed that caveolin, as well as the small GTPase cdc42 were dispensable. Alternatively, internalized GPI-anchored proteins colocalized with flotillin-2-positive vesicles, and down-expression of flotillin-2 inhibited endocytosis of GPI-anchored proteins. These results show that basolateral endocytosis of GPI-anchored proteins in hepatic cells occurs via a clathrin-independent flotillin-dependent pathway. The use of distinct endocytic pathways may explain, at least in part, the different rates of transcytosis between transmembrane and GPI-anchored proteins.