The "metabolic memory", a phenomenon that the target cell remembers the early hyperglycemia, has been reported to be a critical issue in diabetes pathogenesis. Here, we confirmed the inducible effects of high glucose (HG) and HG followed by normal glucose (HN) upon the proliferation and the tube formation capacity of human umbilical vein endothelial cells (HUVECs), as well as the suppressive effects of HG and HN on HUVEC apoptosis. In the meantime, the miR-320 expression could be dramatically downregulated (** and ## P < 0.01), whereas VEGFA expression (** and ## P < 0.01) and VEGFA, PKC, and RAGE protein levels could be remarkably induced via HG and HN stimulation. More importantly, the effects of HG and HN were not significantly different, suggesting the existence of high glucose-induced metabolic memory and the involvement of miR-320 and VEGFA in high glucose-induced metabolic memory in HUVECs. Consistently, miR-320 overexpression significantly reversed the effects of HG and HN on HUVECs (* and # P < 0.05, ** and ## P < 0.01). miR-320 suppressed the expression of VEGFA via direct binding to the 3'-UTR of VEGFA mRNA, therefore suppressing high glucose-induced metabolic memory (** P < 0.01); the effects of miR-320 overexpression on HUVECs could be reversed by VEGFA overexpression (# P < 0.05, ## P < 0.01), indicating that miR-320/VEGFA axis modulates the proliferation, apoptosis, and the angiogenesis capacity of HUVECs. In conclusion, we demonstrate that miR-320/VEGFA axis is crucial to high glucose-induced metabolic memory during HUVEC dysfunction and may be involved in the pathology of diabetes.