Tetrahydrobiopterin (BH4) is an essential cofactor for nitric oxide synthase (NOS). We previously described that hydrogen peroxide (H 2 O 2 ) increases BH4 levels through the induction of GTP-cyclohydrolase I (GTPCH), which is the rate-limiting enzyme for the synthesis of BH4, in vascular endothelial cells. The aim of this study was to examine the underlying mechanism of H 2 O 2 -induced BH4 synthesis in vascular endothelial cells. The increases in BH4 levels induced by H 2 O 2 were strongly reduced by a Janus kinase-2 (Jak2) inhibitor, AG490. The H 2 O 2 -induced increases in GTPCH mRNA expression and GTPCH activity were also blocked by treatment with AG490. H 2 O 2 elicited an increase in the level of phosphorylated Jak2, suggesting that the induction of BH4 by H 2 O 2 was mediated by the Jak2 pathway. Signal transducers and activators of transcription (Stats) are the best-known substrates for Jak2. The H 2 O 2 -induecd increases in BH4 levels were reduced by treatment with fludarabine, which is shown to cause a specific depletion of Stat1 protein but not of other Stats. Moreover, H 2 O 2 caused the DNA binding of Stat1, and this was inhibited by AG490. Stat1 phosphorylation was enhanced by H 2 O 2 treatment, and the phosphorylation was attenuated by AG490. These findings suggest that the stimulation of BH4 synthesis through the induction of GTPCH is mediated at least in-part by the Jak2–Stat1 pathway.