Acid-sensing ion channel (ASIC), an excitatory cation channel gated by extracellular protons, is involved in both central and peripheral neuronal pain pathway. Recently reported African black mamba venom mambalgins are a new class of three-finger peptides that act as pain reducer through the inhibition of ASICs. Through an efficient one-pot chemical synthesis, enough amount of snake venom toxin mambalgin-1 was achieved and exhibited effective biological activity on ASIC1a but not ASIC2a channels. Sequence alignment between ASIC1a and ASIC2a channels reveals residue differences in acid pocket and in turn directs the mutation construction. Electrophysiological experiments suggested an accelerated decay time constance of ASIC1a channel by application of mambalgin-1. Mutations of different residues in the ASIC1a channel acidic pocket exhibited increased or decreased inhibitory effect of the toxin. These data help to understand the binding pattern and inhibition mechanism of mambalgins on ASIC channels.