Acid-sensing ion channel 1a (ASIC1a) is the key proton receptor in nervous systems, mediating acidosis-induced neuronal injury in many neurological disorders, such as ischemic stroke. Up to now, functional ASIC1a has been found exclusively on the plasma membrane. Here we show that ASIC1a proteins are also present in mitochondria of mouse cortical neurons where they are physically associated with adenine nucleotide translocase (ANT). Moreover, purified mitochondria from ASIC1a-/-mice exhibit significantly enhanced Ca2+ retention capacity (CRC) and accelerated Ca2+ uptake rate. When challenged with hydrogen peroxide (H2O2), ASIC1a-/- neurons are resistant to cytochrome C release and inner mitochondrial membrane depolarization, suggesting an impairment of mitochondrial permeability transition (MPT) due to ASIC1a deletion. Consistently, H2O2-induced neuronal death, which is MPT-dependent, is reduced in ASIC1a-/- neurons. Additionally, significant increase in mitochondrial size and oxidative stress level are detected in ASIC1a-/- mouse brain. Furthermore, two-dimensional (2-D) difference gel electrophoresis and mass spectrometry (MS) assays reveal that ten mitochondrial proteins are dramatically changed (> 2 folds) in ASIC1a-/-brains, most of which are closely related to reactive oxygen species (ROS) signal pathways. Thus, our data suggest that mitochondrial ASIC1a may be an important regulator of MPT pore, and contributes to oxidative neuronal cell death.