Endothelial cell (EC) dysfunction is a major symptom associated with the initiation of atherosclerosis (AS). Cyanidin-3-O-glucoside (C3G) has the potentials to attenuate AS symptoms. In the current study, the mechanism driving the effects of C3G on AS rabbits and injured ECs were explored by focusing on the changes in miR-204-5p/SIRT1 axis. AS symptoms were induced in rabbits using high-fatty diet (HFD) plus balloon catheter injured method and handled with C3G of two doses. Then the changes in artery wall structure, hemodynamics parameters, blood lipid level, systemic inflammation, and miR-204-5p/SIRT1 axis were detected. EC dysfunction was imitated by subjecting human umbilical vein endothelial cells (HUVECs) to TNF-α, which was then handled with C3G. The changes in apoptosis, inflammation, and miR-204-5p/SIRT1 axis were detected. The results showed that the administrations of C3G improved artery wall structure and hemodynamics parameters, decreased blood lipid levels, and suppressed pro-inflammatory cytokine production in HFD rabbits, which was associated with the down-regulation of miR-204-5p and the up-regulation of SIRT1. In in vitro assays, the treatments of C3G suppressed apoptosis, inhibited inflammation, down-regulated miR-204-5p level, and induced SIRT1 level in HUVECs. The overexpression of miR-204-5p impaired the protective effects of C3G on the injured HUVECs by increasing cell apoptosis and inflammation. The findings outlined in the current study confirmed the protective effects of C3G on EC function, which was associated with the down-regulation of miR-204-5p and the up-regulation of SIRT1.© 2020 International Union of Biochemistry and Molecular Biology.