Angiogenesis is involved in ischemic heart disease as well as the prognosis of heart failure (HF), and endothelial cells are the main participants in angiogenesis. In this study, we found that miR-221-3p is highly expressed in vascular tissue, especially in endothelial cells, and increased miR-221-3p was observed in heart tissue of HF patients and transverse aortic constriction (TAC)-induced HF mice. To explore the role of miR-221-3p in endothelial cells, microRNA (miRNA) mimics and inhibitors were employed in vitro. Overexpression of miR-221-3p inhibited endothelial cell proliferation, migration, and cord formation in vitro, while inhibition of miR-221-3p showed the opposite effect. Anti-argonaute 2 (Ago2) coimmunoprecipitation, dual-luciferase reporter assay, and western blotting were performed to verify the target of miR-221-3p. Hypoxia-inducible factor-1 alpha (HIF-1 alpha) was identified as a miR-221-3p target, and the adverse effects of miR-221-3p on endothelial cells were alleviated by HIF-1 alpha re-expression. In vivo, a mouse model of hindlimb ischemia (HLI) was developed to demonstrate the effect of miR-221-3p on angiogenesis. AntagomiR-221-3p increased HIF-1 alpha expression and promoted angiogenesis in mouse ischemic hindlimbs. Using the TAC model, we clarified that antagomiR-221-3p improved cardiac function in HF mice by promoting cardiac angiogenesis. Furthermore, serum miR-221-3p was detected to be negatively correlated with heart function in chronic heart failure (CHF) patients. Our results conclude that miR-221-3p inhibits angiogenesis of endothelial cells by targeting HIF-1 alpha and that inhibition of miR-221-3p improves cardiac function of TAC-induced HF mice. Furthermore, miR-221-3p might be a potential prognostic marker of HF. This study describes how miR-221-3p in endothelial cells reduces angiogenesis by inhibiting hypoxia-inducible factor-1 alpha. Because antagonism of miR-221-3p significantly improves the cardiac function of mice with heart failure it may be a new and effective molecular target for progressing and treatment of heart failure.