Phosphatidylcholine transfer protein (PC-TP) is a steroidogenic acute regulatory related transfer (START) domain protein that is enriched in liver cytosol and binds phosphatidylcholines with high specificity. In tissue culture systems, PC-TP promotes ATP binding cassette protein A1-mediated efflux of cholesterol and phosphatidylcholine molecules as nascent pre-beta-HDL particles. Here we explored a role for PC-TP in HDL metabolism in vivo utilizing eight-week old male Pctp^-/- and wild type littermate C57BL/6J mice that were fed for 7 d with either chow or a high fat/high cholesterol diet. In chow fed mice, neither plasma cholesterol concentrations nor the concentrations and compositions of plasma phospholipids were influenced by PC-TP expression. However, in Pctp^-/- mice there was an accumulation of small alpha-migrating HDL particles. This occurred without changes in hepatic expression of ATP binding cassette protein A1 or in proteins that regulate the intravascular metabolism and clearance of HDL particles. In Pctp^-/- mice fed the high fat/high cholesterol diet, HDL particle sizes were normalized, whereas plasma cholesterol and phospholipid concentrations were increased compared with wild type mice. In the absence of upregulation of hepatic ATP binding cassette protein A1, reduced HDL uptake from plasma into livers of Pctp^-/- mice contributed to higher plasma lipid concentrations. These data indicate that PC-TP is not essential for the enrichment of HDL with phosphatidylcholines, but that it does modulate particle size and rates of hepatic clearance.