Niemann-Pick C1-Like 1 (NPC1L1) is expressed in human liver, but its mechanism of action and how it is regulated in this tissue is not well-understood.  Herein, we decided to examine the effects of the inhibitor of NPC1L1, ezetimibe, in the presence and absence of a low cholesterol medium (MITO+ medium), on the expression of NPC1L1 in the human hepatocyte like C3A cell line.  It was demonstrated that ezetimibe and MITO+ medium were able to independently decrease NPC1L1 protein expression while increased the mRNA expression of this transporter.  Treating with ezetimibe and MITO+ medium together additively decreased NPC1L1 protein levels, but their effects on NPC1L1 mRNA expression were counteracting. Cycloheximide studies showed that these treatments enhanced the stability of the NPC1L1 protein in an additive manner, supporting the possibility that the translation efficiency of the NPC1L1 mRNA was decreased under these conditions or that the factors required to degrade the NPC1L1 transporter were inhibited by the presence of cycloheximide.  In addition, the data confirmed that growing the cells in flasks coated with poly-D-lysine (should induce polarization) was required for maximum expression levels of NPC1L1 protein but not for localization of this transporter to the apical membrane.  The information derived from this research is critical to our understanding of the role of hepatic NPC1L1 in removing cholesterol from the bile and in cholesterol homeostasis in general.