Acid-sensing ion channels are proton-gated ion channels that contribute to neuronal responses during acidosis associated with inflammation, tissue injury and ischaemia. The ASIC1 gene produces two major splice variants, ASIC1a and ASIC1b, which differ in their tissue distribution and pharmacology. Venom peptides have provided some of the most potent and mechanistically informative modulators of ASIC channels, including high-affinity inhibitors of ASIC1a that are widely used to investigate channel function and therapeutic potential. In contrast, no peptide inhibitors of human ASIC1b have been established, with known potent peptide modulators instead generally potentiating this channel. To address this gap, we compared the activity of venom peptides across ASIC1a and ASIC1b subtypes from different species, identifying marked differences in potency and mode of action. These findings revealed new opportunities to discover and develop peptides that inhibit human ASIC1b, an important ASIC subtype in peripheral pain. Together, this work highlights the value of venom peptide diversity for uncovering new ASIC pharmacology and expanding the toolkit available to investigate human ASIC1 channels.