Hypoxia-inducible factor-1 alpha (HIF-1 alpha) mediates hypoxic responses and regulates gene expression involved in angiogenesis, invasion and metabolism. Among the various HIF-1 alpha posttranslational modifications, HIF-1 alpha methylation and its physiological role have not yet been elucidated. Here we show that HIF-1 alpha is methylated by SET7/9 methyltransferase, and that lysine-specific demethylase 1 reverses its methylation. The functional consequence of HIF-1 alpha methylation is the modulation of HIF-1 alpha stability primarily in the nucleus, independent of its proline hydroxylation, during long-term hypoxic and normoxic conditions. Knock-in mice bearing a methylation-defective Hif1a(KA/KA) allele exhibit enhanced retinal angiogenesis and tumour vascularization via HIF-1 alpha stabilization. Importantly, S28Y and R30Q mutations of HIF-1 alpha, found in human cancers, are involved in the altered HIF-1 alpha stability. Together, these results demonstrate a role for HIF-1 alpha methylation in regulating protein stability, thereby modulating biological output including retinal and tumour angiogenesis, with therapeutic implications in human cancer.