Supernatant protein factor (SPF) is a novel cholesterol biosynthesis-accelerating protein expressed in liver and small intestine. Here, we report on the physiological role of SPF by using Spf-deficient mice. Although plasma cholesterol levels were similar in chow-fed Spf^-/- and wild-type (WT) mice, fasting significantly decreased plasma cholesterol levels in Spf^-/- mice but not in WT mice. While fasting reduced hepatic cholesterol synthesis rate in WT mice, a more pronounced reduction was observed in Spf^-/- mice. The expression of cholesterogenic enzymes was dramatically suppressed by fasting both in WT and Spf^-/- mice. In contrast, hepatic SPF expression of WT mice was up-regulated by fasting in peroxisome proliferator-activated receptor (PPAR-)-dependent manner. These results indicate that in WT mice, the decrease of hepatic cholesterol synthesis under fasting conditions is at least in part compensated by SPF up-regulation. Fibrates, which function as a PPAR- agonist and are widely used as hypotriglycemic drugs, reduced hepatic cholesterol synthesis and plasma cholesterol levels by approximately one-half in Spf^-/- mice but not in WT mice. These findings suggest that co-administration of fibrates and an SPF inhibitor may reduce not only plasma triglyceride but also cholesterol levels, indicating that SPF is a promising hypocholesterolemic drug target.--Shibata, N., Jishage, K.-i., Arita, M., Watanabe, M., Kawase, Y., Nishikawa, K., Natori, Y., Inoue, H., Shimano, H., Yamada, N., Tsujimoto, M., Arai, H. Regulation of hepatic cholesterol synthesis by a novel protein (SPF) that accelerates cholesterol biosynthesis.