Acid sensing ion channels (ASIC) are voltage insensitive, cationic channels that have recently been identified in vascular smooth muscle (VSM). It is possible that ASIC contribute to vascular reactivity via Na⁺ and Ca²⁺ conductance; however their function in VSM is largely unknown. In pulmonary VSM, store-operated Ca²⁺ entry (SOCE) plays a significant role in vasoregulatory mechanisms such as hypoxic pulmonary vasoconstriction and receptor-mediated arterial constriction. Therefore, we hypothesized that ASIC contribute to SOCE in pulmonary VSM. We examined SOCE resulting from depletion of intracellular Ca²⁺ stores with cyclopiazonic acid in isolated small pulmonary arteries and primary cultured pulmonary arterial smooth muscle cells by measuring 1) changes in VSM [Ca²⁺]_i using fura-2 indicator dye, 2) Mn²⁺ quenching of fura-2 fluorescence, and 3) store-operated Ca²⁺ and Na⁺ currents using conventional whole cell patch-clamp configuration in voltage-clamp mode. The role of ASIC was assessed by the use of the ASIC inhibitors, amiloride, benzamil and psalmotoxin 1, or siRNA directed towards ASIC1, ASIC2, or ASIC3 isoforms. We found that store-operated VSM [Ca²⁺]_i responses, Mn²⁺ influx, and inward cationic currents were attenuated by either pharmacologic ASIC inhibition or treatment with ASIC1 siRNA. These data establish a unique role for ASIC1 in mediating SOCE in pulmonary VSM, and provide new insight into mechanisms of VSM Ca²⁺ entry and pulmonary vasoregulation.