Tissue matrix remodeling and fibrosis leading to loss of pulmonary arterial and right ventricular compliance are important features of both experimental and clinical pulmonary hypertension (PH). We have previously reported that transglutaminase 2 (TG2) is involved in PH development while others have shown it to be a crosslinking enzyme that participates in remodeling of extracellular matrix in fibrotic diseases in general. In the present studies, we used a mouse model of experimental PH (Sugen 5416 and hypoxia; SuHypoxia) and cultured primary human cardiac and pulmonary artery adventitial fibroblasts to evaluate the relationship of TG2 to the processes of fibrosis, protein crosslinking, extracellular matrix collagen accumulation and fibroblast to myofibroblast transformation. We report here that TG2 expression, activity as measured by serotonylated fibronectin and protein crosslinking activity along with fibrogenic markers are significantly elevated in lungs and right ventricles of SuHypoxic mice with PH. Similarly, TG2 expression and activity, protein crosslinking activity and fibrogenic markers are significantly increased in cultured cardiac and pulmonary artery adventitial fibroblasts in response to hypoxia exposure. Pharmacological inhibition of TG2 activity with ERW1041E significantly reduced hypoxia-induced crosslinking activity and synthesis of collagen 1 and α-smooth muscle actin in both the in vivo and in vitro studies. TG2 siRNA had a similar effect in vitro. Our results suggest that TG2 plays an important role in hypoxia-induced pulmonary and right ventricular tissue matrix remodeling in the development of PH.