Objective To explore the role of Piezo2 in venous vascular smooth muscle cell (VSMC) dysfunction and neointimal hyperplasia following exposure of veins to an arterial mechanical environment, and elucidate its potential role in venous restenosis after coronary artery bypass grafting (CABG) and arteriovenous fistula (AVF) surgery. Methods Based on transcriptomic datasets, differentially expressed genes between AVF or grafted veins with normal veins were analyzed using GEO2R and GO. Immunofluorescence was used to detect expression of Piezo2 in two AVF clinical samples. FX-5000TM cyclic stretch application system was used to apply 1.25 Hz stretch with 15% amplitude to VSMCs to simulate arterial conditions in vitro. The protein expressions of Piezo2, SM22 (VSMC phenotypic marker), and PCNA (proliferation-related molecule) were measured with Western Blot. Calcium-free medium was further used to remove extracellular Ca2?, and the effect of Ca2? on stretch-induced changes in related molecules were analyzed. Results Transcriptomic analysis revealed that Piezo2 was upregulated in AVF and grafted veins compared to normal veins. Immunofluorescence showed the increased protein expression of Piezo2 in AVF tissues compared to normal veins. Notably, in the neointimal tissue of AVF samples, Piezo2 was significantly upregulated, while SMA was downregulated. In vitro, 15% cyclic stretch upregulated Piezo2 and PCNA expression but downregulated SM22 expression, which suggested a phenotypic switch of venous VSMCs from the contractile phenotype to the synthetic phenotype. Removal of extracellular Ca2? partially reversed the stretch-induced VSMC phenotypic switch and proliferation. Conclusions After veins exposed to an arterial mechanical environment, the upregulated Piezo2 may induce neointimal hyperplasia by promoting VSMC phenotypic switch. This study may provide mechanobiological insights and potential therapeutic targets for the prevention and treatment of venous restenosis following CABG and AVF surgeries.