To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1) in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposing to hypoxia, a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE in PASMCs were intensified when TRPC1, STIM1 and Orai1 were upregulated. SOCE in cells transfected with specific small hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1 or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1 and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1 and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.