Phosphatidylcholine transfer protein (PC-TP) is a member of the steroidogenic acute regulatory transfer protein-related (START) domain superfamily and is enriched in liver. To explore a role for PC-TP in hepatic cholesterol metabolism, Pctp^-/- and wild type C57BL/6J mice were fed a standard chow diet or a high fat/high cholesterol lithogenic diet. In chow fed Pctp^-/- mice, acyl CoA:cholesterol acyltransferase (Acat) activity was markedly increased, HMG-CoA reductase activity was unchanged and cholesterol 7-hydroxylase (Cyp7A1) activity was reduced. Consistent with increased Acat activity, esterified cholesterol concentrations in livers of Pctp^-/- mice were increased whereas unesterified cholesterol concentrations were reduced. Hepatic phospholipid concentrations were also decreased in the absence of PC-TP and consequently, unesterified cholesterol/phospholipid ratios in liver remained unchanged. The lithogenic diet downregulated HMG-CoA reductase wild type and Pctp^-/- mice, whereas Acat was increased only in wild type mice. In response to the lithogenic diet, a greater reduction in Cyp7A1 activity in Pctp^-/- mice could be attributed to increased size and hydrophobicity of the bile salt pool. Despite higher hepatic phospholipid concentrations, the unesterified cholesterol/phospholipid ratio increased. The lack of Acat upregulation suggests that, in the setting of the dietary challenge, the capacity for esterification to defend against hepatic accumulation of unesterified cholesterol was exceeded in the absence of PC-TP expression. We speculate that regulation of cholesterol homeostasis is a physiological function of PC-TP in liver, which can be overcome with a cholesterolrich lithogenic diet.