Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap ...Gap junctions form intercellular conduits with a large pore size whose closed and open states regulate communication between adjacent cells. The structural basis of the mechanism by which gap junctions close, however, remains uncertain. Here, we show the cryo-electron microscopy structures of innexin-6 (INX-6) gap junction proteins in an undocked hemichannel form. In the nanodisc-reconstituted structure of the wild-type INX-6 hemichannel, flat double-layer densities obstruct the channel pore. Comparison of the hemichannel structures of a wild-type INX-6 in detergent and nanodisc-reconstituted amino-terminal deletion mutant reveals that lipid-mediated amino-terminal rearrangement and pore obstruction occur upon nanodisc reconstitution. Together with molecular dynamics simulations and electrophysiology functional assays, our results provide insight into the closure of the INX-6 hemichannel in a lipid bilayer before docking of two hemichannels.