Sirtuins, a family of NAD(+)-dependent deacetylase enzymes, have been identified as mammalian homologs of yeast silent information regulator 2 (SIR2). Sirtuin 6 (SIRT6) plays important roles in cell homeostasis, DNA damage repair, cancer suppression, and aging. SIRT6 overexpression improves metabolic diseases, such as hypercholesterolemia, cholesterol-related disease, and type 2 diabetes via AMP-activated protein kinase (AMPK) activation. SIRT6 is abundant in the liver and is a crucial target for patients with liver steatosis. Compounds for drug repositioning were screened to identify potential SIRT6 activators, and fluvastatin, a synthetic inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase that reduces cholesterol synthesis, was identified to activate SIRT6. When HepG2 cells were treated with fluvastatin, the expression of SIRT6 and phosphorylation of sterol regulatory element-binding protein (SREBP)-1 and AMPK alpha, which is regulated by SIRT6, increased. In this study, we examined the mechanism underlying cholesterol regulation by fluvastatin via SREBP-1 and AMPK alpha pathway and suggested that fluvastatin is an SIRT6 activator that regulates cholesterol homeostasis and fatty liver disease. (C) 2018 Elsevier Inc. All rights reserved.