Acid-sensing ion channels (ASICs) are sodium channels activated by extracellular protons. ASICs are expressed throughout the peripheral and central nervous system and they are involved in pain perception, synaptic transmission, and neuronal death during ischemia. Psalmotoxin 1 (PcTx1), a peptide toxin isolated from tarantula venom, was reported to potently and specifically inhibit ASIC1a activity. Recently, we have shown that PcTx1 inhibits ASIC1a by increasing its apparent affinity for protons. Here we studied whether PcTx1 also interacts with ASIC1b, a splice variant of -1a. We found that PcTx1 interacts with ASIC1b and shifts the pH activation curves of ASIC1b to more basic pH values. However, PcTx1 did not inhibit ASIC1b but promoted channel opening under slightly acidic conditions. PcTx1 also strongly slowed the desensitization process of ASIC1b, but not of ASIC1a. These data can be best explained by assuming that PcTx1 has different affinities for closed, open, and desensitized states of the channels. For ASIC1b, PcTx1 stabilizes its open state, whereas for ASIC1a, it rather stabilizes the desensitized state. Moreover, we used a series of chimera between ASIC1a and ASIC1b to identify a domain in the extracellular loop that is responsible for the difference of PcTx1's apparent affinity for the desensitized state of ASIC1a and 1b.