Acid-sensing ion channel 3 (ASIC3) is a proton-gated Na⁺ channel that belongs to the DEG/ENaC family of Ion channels. It is expressed in peripheral sensory neurons and plays an important role in nociception. It has been shown that the integral membrane protein stomatin interacts with ASIC3 and inhibits its currents. The majority of stomatin forms a hairpin structure with intracellular N- and C-termini. However, about 10% of the stomatin pool adopts a transmembrane form with an extracellular C terminus carrying five N-glycans. Here we tried to identify the interaction site(s) between ASIC3 and stomatin. We generated chimeric constructs between stomatin and its non-inhibiting human homologue SLP (stomatin like protein) 3 and tested their effects on ASIC3 currents by TEVC in Xenopus oocytes. We found that in addition to the extreme C-terminus, several residues within a central domain of stomatin (aa113–158) are essential for its inhibition. Mutation of glycosylation sites within this central domain of stomatin enhanced inhibition of ASIC3, implicating an interference of glycosylation with the interaction and suggesting the involvement of the transmembrane form. Co-expression of stomatin with ASIC3 did not reduce surface expression of ASIC3 arguing against internalization as the mechanism of ASIC3 inhibition. Mutation of the I₂₆₇VF motif, which is known to be crucial for lipid-raft association of stomatin, abolished the inhibition of ASIC3 currents, suggesting an involvement of lipid rafts. Our results help to molecularly characterize the interaction of stomatin with ASIC3, which may be of physiological relevance in controlling the activity of this ion channel.