Gallbladder epithelial cells (GBEC) are exposed to high and fluctuating concentrations of biliary cholesterol on their apical (AP) surface. GBEC absorb and efflux cholesterol, but the mechanisms of cholesterol uptake, intracellular trafficking and efflux in these cells are not known. We previously reported that ABCA1 mediates basolateral (BL) cholesterol efflux in cultured polarized GBEC. In addition, the nuclear hormone receptors LXR/RXR mediate both AP and BL cholesterol efflux. An interesting finding from our previous study was that apoA-I applied to the AP surfaces of cells elicited BL ABCA1-mediated cholesterol efflux. As ABCA1-mediated cholesterol efflux requires the presence of a cholesterol acceptor, we hypothesized that GBEC synthesize and secrete endogenous apolipoproteins into the BL compartment. Here, we demonstrate that cholesterol loading of cells with model bile and AP apoA-I treatment is associated with an increase in the synthesis of apoE mRNA and protein. Furthermore, apoE is secreted into the BL compartment. LXR/RXR ligands stimulate the synthesis of endogenous apoA-I mRNA and protein, as well as apoE mRNA. BL secretion of apoA-I is elicited by LXR/RXR ligands. Therefore, GBEC synthesize apolipoproteins A-I and E and efflux cholesterol using ABCA1- and non-ABCA1-mediated pathways. These processes may alter gallbladder biliary cholesterol concentrations, and thereby influence gallstone formation.