Induction of diabetes can produce arterial wall hypoxia preceding the formation of vascular lesions. We therefore determined whether a dynamic interplay exists between hyperglycemia and the major regulator of hypoxia, hypoxia-inducible factor 1-alpha (HIF-1α) in controlling hypoxia-induced vascular smooth muscle cell growth in vitro. Bovine aortic smooth muscle cells (BASMC) were exposed to conditions of normal glucose (5.5 mM) and hyperglycemia (25 mM glucose) under normoxic (5% CO₂, 95% air) and hypoxic (2% O₂, 5% CO₂, 93% N₂) conditions for 5 days prior to determining cell proliferation and apoptosis using FACS analysis, immunoblot QRT-PCR and caspase-3 enzymatic activity. Chronic hypoxia stimulated apoptosis and inhibited proliferation in the presence of normal glucose while hyperglycemia significantly attenuated the hypoxic-induced growth response. HIF-1α expression was also inhibited by hyperglycemia with a concomitant decrease in hypoxia response element (HRE) promoter transactivation. Subsequent siRNA knockdown of HIF-1α inhibited the hypoxia-induced changes in growth in the presence of normal glucose while concomitantly attenuating the effects of hyperglycemia on the hypoxic-induced response. These results suggest that hypoxia-induced changes in vascular cell growth are altered by hyperglycemia via inhibition of HIF-1α expression and activity.