Endothelial progenitor cells (EPCs) are vital to the recovery of endothelial function and maintenance of vascular homeostasis. EPCs mobilize to sites of vessel injury and differentiate into mature endothelial cells (ECs). Locally mobilized EPCs are exposed to cyclic stretch caused by blood flow, which is important for EPC differentiation. MicroRNAs (miRNAs) have emerged as key regulators of several cellular processes. However, the role of miRNAs in cyclic stretch-induced EPC differentiation remains unclear. Here, we investigate the effects of microRNA-129-1-3p (miR-129-1-3p) and its novel target Runt-related transcription factor 2 (Runx2) on EPC differentiation induced by cyclic stretch. Bone marrow-derived EPCs were exposed to cyclic stretch with a magnitude of 5% (which mimics physiological mechanical stress) at a constant frequency of 1.25 Hz for 24 hours. The results from a miRNA array revealed that cyclic stretch significantly decreased miR-129-1-3p expression. Furthermore, we found that downregulation of miR-129-1-3p during cyclic stretch-induced EPC differentiation toward ECs. Meanwhile, expression of Runx2, a putative target gene of miR-129-1-3p, was increased as a result of cyclic stretch. A 3'UTR reporter assay validated Runx2 as a direct target of miR-129-1-3p. Furthermore, small interfering RNA (siRNA)-mediated knockdown of Runx2 inhibited EPC differentiation into ECs and attenuated EPC tube formation via modulation of vascular endothelial growth factor (VEGF) secretion from EPCs in vitro. Our findings demonstrated that cyclic stretch suppresses miR-129-1-3p expression, which in turn activates Runx2 and VEGF to promote endothelial differentiation of EPCs and angiogenesis. Therefore, targeting miR-129-1-3p and Runx2 may be a potential therapeutic strategy for treating vessel injury.