The receptor tyrosine kinase Axl is involved in diabetic vascular disease. This study aims to investigate the effect of high glucose on endothelial cells injury and Axl expression in hypoxia condition in vitro, and we present details of the mechanism associated with overexpression of Axl rescue the high glucose injury. Our results showed that high glucose impaired both human umbilical vein endothelial cells (HUVECs) and EAhy926 cells angiogenesis in hypoxia condition. In addition, high glucose inhibits Axl and hypoxia-inducible factor 1- (HIF-1) protein expression in hypoxia condition. Axl overexpression significantly reversed endothelial cells dysfunction in high glucose/hypoxia. Furthermore, Axl overexpression in EAhy926 cells increases HIF-1 protein synthesis through PI3K/Akt/mTOR/p70 (S6K) signal pathway but not Mek/Erk in high glucose/hypoxia condition. This study demonstrates that high glucose can alter Axl signaling and HIF-1 in hypoxia condition. Overexpression of Axl may rescue endothelial cells dysfunction and HIF-1 expression through its downstream signals in high glucose/hypoxia.