Purpose: Exercise has cardiovascular benefits which might be related to endothelial progenitor cells (EPC). Meanwhile, there is evidence suggesting that EPC-derived exosomes (EPC-EX) promote vascular repair and angiogenesis through their carried microRNA (miR)-126. In this study, we investigated whether exercise could increase the levels of circulating EPC-EX and their miR-126 cargo, and by which promote the protective function of EPC-EX on endothelial cells (EC). Methods: Plasma EPC-EX from sedentary, low, or moderate exercise mice, respectively, denoted as EPC-EXS, EPC-EXL, and EPC-EXM, were isolated using microbead-based sorting techniques and characterized by nanoparticle tracking analysis, Western blot, and quantitative real-time polymerase chain reaction assessments of biomarkers and miR-126. High glucose (25 mM) with hypoxia (1% O2) was used for inducing an EC injury model. The injured EC were treated by coculturing with vehicle, EPC-EXS, EPC-EXL, EPC-EXM, or EPC-EXM + anti–miR-126. After that, EC were used for flow cytometry analysis of apoptosis, assessments of tube formation and migration, and measurements of miR-126 level and its downstream sprouty-related protein-1 (SPRED1) and vascular endothelial growth factor (VEGF). Results: 1) Isolated EPC-EX positively expressed exosomal markers (CD63 and Tsg101) and EPC markers (CD34 and VEGFR2). 2) Exercise intensity dependently elevated plasma level of EPC, EPC-EX/EPC ratio, and miR-126 expression in EPC and EPC-EX. 3) Injured EC displayed apoptosis increment, angiogenic dysfunction and miR-126 reduction. 4) EPC-EXM had better effects than EPC-EXS and EPC-EXL on alleviating those changes of injured EC, accompanied with SPRED1 downregulation and VEGF upregulation. 5) The effects of EPC-EXM were abolished by miR-126 knockdown. Conclusions: Our data demonstrate that exercise can increase EPC-EX release and miR-126 level and enhance the effects of EPC-EX on protecting EC against injury through the SPRED1/VEGF pathway.