Acid-Sensing Ion Channels (ASICs) are gated by extracellular protons and play important roles in physiological and pathological states, such as pain and stroke. ASIC1a and ASIC2a, two of the most highly expressed subunits in the brain, form functional homo- and hetero-meric (ASIC1a/2a) channels. The function of ASIC1a has been widely studied using psalmotoxin (PcTx1), a venom-derived peptide, as an ASIC1a-selective antagonist. Here, using whole-cell patch clamp, we show that PcTx1 has dual actions at ASIC1a/2a. It can either inhibit or potentiate the heteromeric channel, depending on the conditioning and stimulating pHs. Potent inhibition occurs only at conditioning pHs that begin to desensitize the channel (IC50 = 2.9 nM at pH7.0, a threshold pH for desensitization of ASIC1a/2a). By contrast, potent potentiation can occur at the physiological pH in both CHO cells (EC50 = 56.1 nM) and cortical neurons (threshold concentration < 10 nM). PcTx1 potentiates ASIC1a/2a by increasing the apparent affinity of channel activation for protons. As such, potentiation is the strongest at moderate pHs, diminishing with increasing proton concentrations. Our findings identify PcTx1 as a valuable tool for studying ASIC1a/2a function and contribute significantly to the understanding of the diverse and complex pharmacology of PcTx1.