Gap junction plaques (GJPs) enable direct intercellular communication and consist of connexin channels arranged into two-dimensional lattices. While structures of purified connexin channels have informed models of gating, they omit key intracellular regions and lack native context. Here, we use cryo-electron tomography and focused ion beam milling to determine the in situ structure of human connexin 43 (Cx43) GJPs in HEK293 cells at 14-Å resolution. We reveal a previously unresolved structural contribution of the large carboxyl-terminal domain to lateral channel-channel interactions that appear critical for plaque assembly. Coarse-grained molecular dynamics simulations suggest how lipids and cholesterol occupy the space between adjacent connexins. These findings resolve a decades-old question regarding gap junction organization and highlight a mechanistic function for the carboxyl-terminal domain, likely regulated by a helix-loop-helix motif. Our study provides a structural blueprint for understanding how connexin diversity and regulation shape tissue-level communication in health and disease.