Objective To investigate the role of microRNA-214-3p (miR-214-3p) in differentiation and proliferation of endothelial progenitor cells (EPCs) induced by cyclic stretch. Methods EPCs were exposed to cyclic stretch at physiological level (with the magnitude of 5%, at a constant frequency of 1.25 Hz) for 24 h by FX-5000T Strain Unit. miRNAs array was performed to identify the expression profiling of miRNAs. Real-time PCR was used to examine the expression levels of miRs. The expression of vascular smooth muscle cells (VSMCs) markers in EPCs was detected by real-time PCR. EPC proliferation was detected by BrdU ELISA assay. After EPCs were transfected with miR-214-3p inhibitor (IN) to knockdown expression of miR-214-3p, the level of VSMC markers expression and EPC proliferation was detected. Results Cyclic stretch significantly decreased miR-214-3p expression, depressed EPC differentiation toward VSMCs, and increased EPCs proliferation. Similarly, transfection with the miR-214-3p inhibitor led to the decreased expression of VSMC markers under static station. Meanwhile, miR-214-3p down-regulation promoted EPC proliferation significantly. Conclusions Physiological cyclic stretch could down-regulate the expression of miR-214-3p in EPCs, depress EPC differentiation towards VSMC and promote EPC proliferation eventually. Therefore, the research findings provide a potential therapeutic strategy for treating vessel injuries.