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Endothelial-to-mesenchymal transition (EndMT) contribute to diabetic cardiac fibrosis, the underlying mechanisms are poorly understood. In the study, we aimed to investigate the role of miR-328 in EndMT mediated by high glucose (HG) and the signaling pathways implicated in human umbilical vein endothelial cells (HUVECs).EndMT of HUVECs was determined by immunofluorescent staining and western blot of the markers CD31 and α-SMA. Real-time polymerase chain reaction was used to detect mRNA expression of miR-328 and transforming growth factor β1 (TGF-β1). SB431542 was used to study the relation of miR-328 and TGF-β1 during EndMT induced by HG. Over-expression and inhibition of miR-328 were achieved by transduction of miR-328 and antagomiR-328. The effects of miR-328 on expression of type I and III collagen, p-MEK1/2, p-ERK1/2 were examined by Western blot.The level of miR-328 was significantly up-regulated in HG-induced EndMT. MiR-328 showed the independent capability of inducing EndMT, which was not related to TGF-β1, and this effect was abrogated by antagomiR-328. MiR-328 affected type I collagen in a time- and dose-dependent manner and enhanced protein expression of type I and III collagens. Further investigation displayed that a significantly higher expression of p-MEK1/2 and p-ERK1/2 in HUVECs transduced with miR-328, and a lower expression of p-MEK1/2 and p-ERK1/2 in cells transduced with antagomiR-328.These results suggest a novel role for miR-328 in HG-induced EndMT, MEK1/2-ERK1/2 pathway is likely to be involved in the associated effects. Our findings may suggest antagomiR-328 as an alternative agent in prevention of HG-induced EndMT.