Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease, characterized by abnormal muscularization and endothelial dysfunction in pulmonary artery (PA). Previous study showed that endothelial extracellular signal-regulated kinase 2 (eERK2) released vasoconstrictive factors and decreased NO bioactivity resulting in obese-induced hypertension and endothelial dysfunction. However, the role of eERK2 in the pathogenesis of PAH remains to be elucidated. Hypothesis: We hypothesized eERK2 impaired eNOS function and progressed the pathogenesis of PAH. Methods and Results: We generated eERK2 knock out mice (EE2KO) crossing Tie2-Cre mice and ERK2 flox mice and induced PAH by exposing mice to chronic hypoxia (10%) for 4 weeks. The littermate ERK2 flox mice without Cre expression were used as Control (Ctr). Immuno-histochemical staining showed the deficiency of eERK2 expression in the PA of EE2KO. We assessed the medial thickness of pulmonary arteries (MTPA) and measured right ventricular systolic pressure (RVSP). Although Ctr exhibited the progression of PA muscularization with an increase in MTPA, EE2KO revealed the significantly attenuated MTPA under hypoxic condition. Moreover, we found the lower RVSPs in EE2KO than those in Ctr under hypoxia in spite of similar RVSPs in normoxia. In RT-PCR, lung endothelin1 (ET1) gene expression was significantly lower in EE2KO than Ctr under hypoxia. Interestingly, hypoxia increased eNOS protein expression both in Ctr and EE2KO with Western blot analysis. The phosphorylation of AMPKα and eNOS were up-regulated in the lungs of EE2KO relative to Ctr under hypoxia. In addition, eNOS dimer/monomer ratios with non redusing SDS-PAGE was significantly increased in EE2KO compared to the Ctr under hypoxia (1.29±0.10 vs. 0.87±0.02, P=0.02, dimer / monomer ratios), suggesting improved eNOS uncoupling. Conclusions: These findings suggest that hypoxia increased eNOS expression and function, and that eERK2 impaired eNOS function by decreasing the phosphorylation of AMPKα and eNOS, and inducing eNOS uncoupling. In addition, ET1 synthesis through eERK2 might contribute to these pathogenesis of PAH with hypoxia.