Transient receptor potential vanilloid 4 (TRPV4) is a mechanosensitive channel in pulmonary arterial smooth muscle cells (PASMCs). Its upregulation by chronic hypoxia is associated with enhanced myogenic tone and genetic deletion of trpv4 suppresses the development of chronic hypoxic pulmonary hypertension (CHPH). Here we further examine the roles of TRPV4 in agonist-induced pulmonary vasoconstriction and in the enhanced vasoreactivity in CHPH. Initial evaluation of TRPV4-selective antagonists HC-067047 and RN-1734 in KCl-contracted PAs of trpv4^-/- mice found that submicromolar HC-067047 was devoid of off-target effect on pulmonary vasoconstriction. Inhibition of TRPV4 with 0.5 µM HC-067047 significant reduced the sensitivity of serotonin (5-HT) induced contraction in WT PAs, but had no effect on endothelin-1 or phenylephrine-activated response. Similar shift in the concentration-response curve of 5-HT was observed in trpv4^-/- PAs, confirming specific TRPV4 contribution to 5-HT-induced vasoconstriction. 5-HT-induced Ca²⁺ response was attenuated by HC-067047 in WT PASMCs but not in trpv4^-/- PASMCs, suggesting TRPV4 is a major Ca²⁺ pathway for 5-HT-induced Ca²⁺ mobilization. Chronic exposure (3-4 weeks) of WT mice to 10% O₂ caused significant increase in 5-HT-induced maximal contraction, which was partially reversed by HC-067047. In concordance, the enhancement of 5-HT-induced contraction was significantly reduced in PAs of CH trpv4^-/- mice. These results suggest unequivocally that TRPV4 contributes to 5-HT-dependent pharmco-mechanical coupling, and plays a major role in the enhanced pulmonary vasoreactivity to 5-HT in CHPH. Since TRPV4 participates in multiple pathological changes in CHPH, it can be considered as a potential target for the treatment of pulmonary hypertension.