Background and Purpose: In diabetes, elevated blood sugar (BS) levels contribute to the overproduction of reactive oxygen species (ROS), leading to endothelial progenitor cell (EPC) dysfunction. 2-hydroxy hispolon (2HH), a derivative of Hispolon (HIS), shows potential in reversing high glucose-induced EPC dysfunction. Experimental Approach: In vitro, we inspected the effect of 2HH on three different angiogenic cells, including outgrowth endothelial cell (OEC), circulating angiogenic cell (CAC) and endothelial cell (EC). In vivo, High-fat diet and streptozotocin induced diabetic mice and hindlimb ischemic model were utilized to evaluate the effect of 2HH on angiogenesis and CAC mobilization. Key Results: Treatment with 2HH significantly improved the proliferation, migration, and tube formation, NO synthesis and ROS diminishing of OEC under high glucose conditions. Moreover, 2HH administration potentiates the AMP-activated protein kinase (AMPK)/heme oxygenase-1 (HO-1) and phosphoinositide 3-kinases (PI3K)/protein kinase B (AKT)/endothelial nitric oxide synthase (eNOS) signaling pathways in OECs exposed to high glucose levels. Additionally, 2HH reverses high glucose-induced impairment of CAC function but fails to restore EC dysfunction under high glucose conditions. Hindlimb ischemic assay demonstrated that 2HH treatment enhances blood flow recovery in ischemic hindlimbs and increases the number of circulating CACs in diabetic mice after femoral ligation. Conclusion and Implications: Our study has demonstrated that 2HH effectively reduces oxidative stress, thereby preventing EPC dysfunction induced by high glucose levels, primarily through the PI3K/AKT/eNOS and AMPK/HO-1 pathways. These findings offer a promising therapeutic avenue for attenuating the susceptibility to critical limb ischemia in patients with diabetes.