Capsaicin is a pungent alkaloid abundantly present in peppers with outstanding biological activities, including the anti-atherosclerosis effect. Previous studies revealed that gut microbiota played an important role in the beneficial effects of capsaicin, but whether it is essential for the anti-atherosclerosis effect of capsaicin is unclear. This study evaluated the anti-atherosclerosis effect of capsaicin in ApoE−/− mice and further explored the role of depleting gut microbiota in the improvement of atherosclerosis. The results showed that capsaicin administration could prevent the development of atherosclerosis and improve serum lipids and inflammation, while antibiotic intervention abolished the alleviation of atherosclerosis by capsaicin. In addition, capsaicin administration could significantly increase the abundance of Turicibacter, Odoribacter, and Ileibacterium in feces, and decrease the abundance of deoxycholic acid, cholic acid, hypoxanthine, and stercobilin in cecal content. Our study provides evidence that gut microbiota plays a critical role in the anti-atherosclerosis effect of capsaicin. © 2022 by the authors.