Sustained pulmonary vasoconstriction and excessive pulmonary vascular wall thickening are two major causes for elevated pulmonary vascular resistance and pulmonary arterial pressure in patients with pulmonary arterial hypertension. Chloroquine, as an anti‐malaria agent, is found to have inhibitory effects on autophagy and a relaxant effect on airway smooth muscle cells. In this study, we observed that chloroquine (100–200 μM) significantly attenuates agonist‐ and high K+‐induced contraction in isolated rat pulmonary artery (PA). Pretreatment with L‐NAME or indomethacin and functional removal of endothelium failed to inhibit chloroquine‐induced relaxation in isolated PA rings pre‐constricted with phenylephrine or 40 mM K+. In pulmonary arterial smooth muscle cells (PASMCs), extracellular application of chloroquine significantly attenuated store‐operated Ca2+ entry induced by passive depletion of intracellularly stored Ca2+ in the sarcoplasmic reticulum using cyclopiazonic acid and partially inhibited ATP‐induced Ca2+ entry. Furthermore, chloroquine was shown to effectively attenuate Ca2+ entry via voltage‐dependent Ca2+ channels (VDCC). In vivo experiments showed that 50mg/kg chloroquine treatment (ip injections) significantly attenuated the development of pulmonary hypertension in rats exposed to chronic hypoxia (10% O2 for 3 weeks). These data indicate that chloroquine is a potent vasodilator of PA and a potential blocker of VDCC, store‐operated Ca2+ channels and receptor‐operated Ca2+ channels in PASMCs. The therapeutic potential of chloroquine on pulmonary hypertension is likely due to the combination of its vasodilative, anti‐proliferative and anti‐autophagic effects.Support or Funding InformationThis work was supported in part by the grants from the National Natural Science Foundation of China (81630004, 81470246, 81220108001, 81520108001), Guangzhou Department of Education Yangcheng Scholarship (12A001S), and Guangdong Province Universities, Colleges Pearl River Scholar Funded Scheme of China and China Scholarship Council (201608440324), as well as from the National Heart, Lung and Blood Institute of the National Institutes of Health (HL115014, HL066012).