Hyperglycaemia and hypoxia play essential pathophysiological roles in diabetes. We determined whether hyperglycaemia influences endothelial cell growth under hypoxic conditions in vitro. Using a Ruskinn Invivo2 400 Hypoxia Workstation, bovine aortic endothelial cells (BAEC) were exposed to high glucose concentrations (25 mM glucose) under normoxic or hypoxic conditions before cell growth (balance of proliferation and apoptosis) was assessed by fluorescence-activated cell sorting (FACS) analysis, proliferating cell nuclear antigen (pCNA), Bcl-xL and caspase-3 protein expression and activity. Hypoxia increased hypoxia response element (HRE) transactivation and induced hypoxia-inducible factor-1α (HIF-1α) expression when compared to normoxic controls concomitant with a significant decrease in cell growth. High glucose (25 mM) concentrations attenuated HRE transactivation and HIF-1α protein expression while concurrently reducing hypoxia-induced changes in BAEC growth. Knockdown of HIF-1α expression significantly decreased hypoxia-induced changes in growth and attenuated the modulatory effects of glucose. These results provide evidence that hypoxia-induced control of BAEC growth can be altered by the presence of glucose via inhibition of HIF-1α expression and activation.