AG490 is a tyrphostin originally described as a Janus Activated Kinase (JAK) 2 inhibitor. AG490 also inhibits epidermal growth factor receptor (EGFR) and guanylyl cyclases (GC). More recently, AG490 was associated with oxidative stress protection in experimental acute kidney injury models. We now show that AG490 is also a strong activator of the Hypoxia Inducible Factor (HIF)-1. Under normoxic conditions HIF-1α is degraded through hydroxylation, von Hippel Lindau protein (VHL)-mediated ubiquitin tagging and proteasomal degradation. AG490 increased HIF-1α protein, but not HIF-1α mRNA levels, dose- and time-dependently in cultured endothelial, vascular smooth muscle and kidney proximal tubular epithelial cells. AG490 increased HIF-1α protein half-life, suggesting that HIF-1α protein accumulation resulted from a decreased degradation. In this regard, AG490 prevented HIF-1α hydroxylation and increased HIF-1α protein levels in human renal carcinoma cells expressing VHL, but did not further increase HIF-1α in VHL negative cells. AG490 did not prevent the proteasomal degradation of other proteins. HIF-1α was not upregulated by dominant negative JAK2constructs, tyrphostin AG9, the EGFR inhibitors erbstatin and genistein, the GC inhibitor Ly83583 or cGMP analogues. Finally, AG490 also increased HIF-1α transcriptional activity evidenced by the increased HIF-1α-dependent VEGF expression. In conclusion, AG490 is a novel HIF-1α activator that increases HIF-1α half-life and protein levels through interference with HIF-1α hydroxylation and VHL-mediated degradation. This action may contribute to the cell and tissue protective effects of AG490.