Gut microbiota plays an important role in many metabolic diseases and has been linked to cardiovascular disease, including atherosclerosis. Clinical studies suggest that red yeast rice (RYR) has the potential to reduce blood lipid levels. However, the mechanisms under which RYR regulates atherosclerosis by affecting the composition of the gut microbiome have not been elucidated. In the current study, results showed that treatment with RYR significantly decreased the plaque formation and levels of cholesterol and low-density lipoprotein (LDL) compared with the atherosclerotic model group which were fed with a high-fat diet. In addition, the height of enteral villus in the red Monascus group was increased, indicating that RYR can improve the intestinal barrier function. Further analysis revealed that RYR might attenuate atherosclerosis through inhibiting hydroxy methylglutaryl-CoA reductase and the consequent inflammatory signaling pathways mediated by TLR2 and TLR4. Moreover, the RYR treatment led to significant structural changes on the intestinal microbiota of high-fat diet-fed mice and reduced the relative abundance of Alistipes and Flavonifractor that exhibited positive relationships with the plasma levels of cholesterol and LDL. Collectively, these findings illustrated that RYR could significantly protect against atherosclerosis, which was possibly associated with the alterations in the gut microbiota composition.