Rationale: Elevated asymmetric dimethylarginine (ADMA) levels contribute to the pathogenesis of hypoxia-induced pulmonary hypertension. Chronic hypoxia decreases the activity of the enzymes metabolising ADMA, dimethylarginine dimethylaminohydrolases (DDAH1 and DDAH2) but the mechanisms responsible are not understood. Objective: To elucidate the physiological significance and the mechanism of hypoxia-induced downregulation of ADMA metabolism. Methods and Results: Exposure of human pulmonary artery endothelial cells (HPAECs) to hypoxia inhibited both DDAH1 and DDAH2 gene and protein expression, reduced DDAH activity and increased ADMA levels. In contrast, in human pulmonary artery smooth muscle cells (HPASMCs) only DDAH2 was reduced while ADMA levels remained unchanged. This endothelium-specific regulation of DDAH1 and ADMA resulted from NFκB-dependent, microRNA-21 (miR-21)-mediated degradation of DDAH1 mRNA. Down regulation of DDAH1 activity contributed to hypoxia-induced endothelial barrier dysfunction and HPASMC proliferation and was prevented by overexpression of DDAH1 and miR-21 blockade. Conversely, overexpression of miR-21 mimicked the effects of hypoxia. DDAH1 overexpressing transgenic mice exposed to 2 weeks hypoxia, showed attenuated pulmonary hypertension and vascular remodelling, compared with wildtype controls. Importantly, inhibition of miR21 in vivo prevented the hypoxia-induced reduction in pulmonary DDAH1 expression and attenuated the development of pulmonary hypertension. Lung tissue samples from hypoxic mice and treatment-naïve IPAH patients also showed reduced DDAH1 expression and increased miR-21 levels, compared with controls. Conclusion: Down regulation of DDAH1 expression by miR-21 in the pulmonary vascular endothelium has a key role in the pathogenesis of hypoxia-induced pulmonary hypertension and may be of broader significance in pulmonary hypertension.