Robinetinidol-(4β,2')-tetrahydroxy-flavone (RBF) is an oligomeric condensed polyphenol that has been shown to exhibit anti-obesity effects in mice. However, little is know regarding its effect on cholesterol synthesis. The present study therefore aimed to investigate the effect of RBF on cholesterol synthesis. It was determined that RBF decreased serum total cholesterol and low density lipoprotein cholesterol in rats by 25.9 and 50.8%, respectively (P<0.001). These results strengthen evidence for the hypothesis that RBF exerts anti-atherogenic effects in vivo. Furthermore, RBF decreased cholesterol synthesis by 72%, when measured using a 3 h period of radiolabeled acetate incorporation into cholesterol, but not when using radiolabelled mevalonate, suggesting that RBF-mediated inhibition occurred largely at or above the level of 3-hydroxy-3-methylglutaryl-coenzymeA (HMG-CoA) reductase. The mechanism by which RBF inactivates HMG-CoA reductase may be attributed to the induction of phosphorylation of adenosine monophosphate (AMP)-kinase, since these results showed that RBF increased phosphorylation of AMP-kinase and HMG-CoA reductase by 2.1- and 3.2-fold, respectively, within 30 min of addition. These results suggest that RBF may be a potential therapeutic agent for hypercholesteremia.