We present the first structure of the human Kir2.1 channel containing both transmembrane domain (TMD) and cytoplasmic domain (CTD). Kir2.1 channels are strongly inward-rectifying potassium channels that play a key role in maintaining resting membrane potential. Their gating is modulated by phosphatidylinositol 4,5-bisphosphate (PIP ₂ ). Genetically inherited defects in Kir2.1 channels are responsible for several rare human diseases, including Andersen’s syndrome. The structural analysis (cryo–electron microscopy), surface plasmon resonance, and electrophysiological experiments revealed a well-connected network of interactions between the PIP ₂ -binding site and the G-loop through residues R312 and H221. In addition, molecular dynamics simulations and normal mode analysis showed the intrinsic tendency of the CTD to tether to the TMD and a movement of the secondary anionic binding site to the membrane even without PIP ₂ . Our results revealed structural features unique to human Kir2.1 and provided insights into the connection between G-loop and gating and the pathological mechanisms associated with this channel.