###### Abstract  
We aimed to examine the effect of natto and red yeast rice (NR) supplementation on lipid and lipoprotein profiles, gene expressions of cholesterol metabolism, and the composition of gut microbiota in ApoE/- mice. Forty-one male ApoE/- mice aged 7-8 wks old were randomly fed a control diet (CD), CD + NR (oral gavage at 0.3 g/kg BW/day), high-fat and high-cholesterol diet (HFD), or HFD + NR for 12 wks. Fasting blood samples, liver and intestine tissues and fecal samples were collected at week 12. Biochemical parameters, gene expressions in cholesterol metabolism and gut microbiota composition and diversity were measured using standard methods. NR supplementation had no significant effect on lipid and lipoprotein profiles. Compared with the HFD group, HFD + NR resulted in higher mRNA expressions of HMGCR and CYPA1 (both _P_-_NR_ \(<\) 0.05) and ABCA1 (_P_-_diet_*NR_ \(=\) 0.0134, _P_-_NR_ \(=\) 0.0407), lower mRNA expression of PCSK9 (_P_-_diet_*NR_ \(=\) 0.0002), lower fasting glucose concentrations (_P_-_diet_*NR_ \(=\) 0.0011), and lower relative abundance of genera _Bacteroides_ and _Lactococcus_ (both _P_-_NR_ \(<\) 0.01) and _Croobacteriaceae_,_LUGG_-_02 (_P_-_diet_*NR_ \(=\) 0.0007). The relative abundance of _Lactococcus_ was inversely correlated with HMGCR and CYPA1, and the relative abundance of _Croobacteriaceae_,_LUGG_-_002_ was positively correlated with PCSK9 and inversely correlated with ABCA1 (all \(P\) \(<\) 0.05). These findings suggest that NR supplementation may regulate gene expressions in cholesterol metabolism via changes in the gut microbiota in HFD-fed ApoE/- mice. nutriscottate; red yeast rice; gene expression; cholesterol metabolism; gut microbiota 2023159733390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.3390/10.390/10.3390/10.3390/10.3390/10.3390/1 Natto, a fermented food mainly consumed in Asian countries, is produced from soybeans through fermentation by _B. subtilis natto_[7]. Nattokinase (NK), which is the most active ingredient in natto, is a potent fibrinolytic enzyme demonstrating beneficial effects on cardiovascular health [8; 9]. Previous studies have reported that the consumption of natto for 2-7 weeks reduces circulating TC or LDL-C concentrations in both human subjects [10] or experimental animals [11; 12]. However, the mechanism underlying the cholesterol-lowering effect of natto has not been reported. Red yeast rice (RYR), which is also a fermented food mainly consumed in Asian countries, is produced by the fermentation of rice by _Monascus purpureus Went_, a species of the mound [13; 14]. RYR contains a bioactive substance called monacolin K (lovastatin), which inhibits 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the pathway of cholesterol synthesis together with other monacolins in RYR [6]. Furthermore, RYR contains phytosterols, fiber and niacin, which collectively may exert cholesterol-lowering effects [15]. Several studies, including animal studies, randomized controlled trials and meta-analyses, have reported the beneficial effects of RYR on reducing circulating TC or LDL-C concentrations [16; 17; 18; 19; 20]. In addition, RYR and its bioactive components have been reported to downregulate the gene expressions of HMG-CoA reductase [19] and Niemann-Pick-C1-Like 1 (NPC1L1) [20] and upregulate the gene expressions of LDL receptor (LDLR) and Cholesterol 7-alpha hydroxylase (CYPA1) [13; 20], indicating that they may decrease circulating TC and LDL-C concentrations via the inhibition of cholesterol synthesis and the absorption and promotion of hepatic cholesterol uptake and bile acid synthesis. In addition to the traditional mechanisms regulating cholesterol metabolism, gut microbiota has been reported to influence circulating cholesterol concentrations through their role in bile acid metabolism and the generation of microbial metabolites, such as short-chain fatty acids (SCFAs) and trimethylamine-N-oxide (TMAO) [21; 22; 23; 24].