Ivermectin (IVM) is a widely used antiparasitic drug in humans and pets which activates glutamate‐gated Cl− channel in parasites. It is also known that IVM binds to the transmembrane domains (TMs) of several ligand‐gated channels, such as Cys‐loop receptors and P2X receptors. In this study, we found that the G‐protein‐gated inwardly rectifying K+ (GIRK) channel is activated by IVM directly. By electrophysiological recordings in Xenopus oocytes, we observed that IVM activates GIRK channel in a phosphatidylinositol‐4,5‐biphosphate (PIP2)‐dependent manner, and that the IVM‐mediated GIRK activation is independent of Gβγ. We found that IVM activates GIRK2 more efficiently than GIRK4. In cultured hippocampal neurons, we also observed that IVM activates native GIRK current. By chimeric and mutagenesis analyses, we identified a unique amino acid residue of GIRK2 among GIRK family, Ile82, located in the slide helix between the TM1 and the N‐terminal cytoplasmic tail domain (CTD), which is critical for the activation. The results demonstrate that the TM‐CTD interface in GIRK channel, rather than the TMs, governs IVM‐mediated activation. These findings provide us with novel insights on the action mode of IVM in ion channels, and information toward identification of new pharmacophores which activate GIRK channel. This article is protected by copyright. All rights reserved