Aim To investigate the role of miR-221 in homocysteine (Hcy)-induced injury of human coronary artery endothelial cells (HCAEC) mediated by cyclin D1. Methods HCAECs were cultured and divided into four groups. The control group was treated with serum-free medium, Hcy group was treated with medium containing 1 mmol/L Hcy, Hcy＋NC (negative control) group was treated with medium containing 1 mmol/L Hcy after transfection with NC inhibitor, and Hcy＋miR-221 group was treated with medium containing 1 mmol/L Hcy after transfection with miR-221 inhibitor. Fluorescence quantitative PCR was used to detect the expression of miR-221, Western blot was used to detect the expression of cyclin D1, MTS was used to detect the OD₄₉₀ nm level of cell viability, flow cytometry was used to detect cell cycle, and double luciferase reporter gene experiment was used to verify miR-221 targeting cyclin D1. Results Compared with the control group, the expression level of miR-221 and the proportion of G0/G1 phase of HCAEC were significantly increased, while the level of OD₄₉₀ nm, the proportion of S phase and G2/M phase and cyclin D1 expression level were significantly decreased in Hcy group. Compared with Hcy group and Hcy＋NC group, the expression level of miR-221 and the proportion of G0/G1 phase of HCAEC were significantly decreased, while the level of OD₄₉₀ nm, the proportion of S phase and G2/M phase and cyclin D1 expression level were significantly increased in Hcy＋miR-221 group. The 1224-1231 base of mRNA 3′UTR of cyclin D1 gene was the binding site of miR-221, and miR-221 reduced the fluorescence activity of wild-type cyclin D1 double luciferase reporter gene. Conclusions The expression of miR-221 increases during Hcy-induced HCAEC injury, and inhibition of miR-221 expression can reduce Hcy-induced HCAEC injury. Targeting cyclin D1 is a possible molecular mechanism.