AbstractGap junction channels, once clustered into gap junction plaques, allow communication of essential metabolites between cells. Gap junction plaques have been reported to be lost from the cell surface during cell division. The mechanism involved in this loss of gap junction plaques during mitosis is unclear, but we hypothesize that an endoexocytotic mechanism that results in cytoplasmic double‐membraned annular gap junction vesicles is involved. In this study, gap junction plaque changes in dividing cells were examined in SW‐13 adrenocortical tumor cells. Endogenous gap junction protein, connexin 43 (Cx43), was detected with immunofluorescence, and live cell imaging was used to monitor green fluorescent protein‐tagged Cx43 (Cx43‐GFP). Mitotic stages were identified by Hoechst chromosomal staining. During interphase, large gap junction plaques were detected; however, the presence of these plaques decreased, whereas cytoplasmic puncta increased beginning with prophase. The cytoplasmic puncta were demonstrated with immunoelectron microscopy to be Cx43‐ positive annular gap junction vesicles. As gap junction plaques reformed at cleavage furrows between daughter cells, the number of annular gap junctions decreased during cytokinesis. The data are consistent with the mechanism of gap junction plaque loss during mitosis relying on an endoexocytotic process that results in annular gap junction vesicles formation. The rapid formation of gap junction plaques during cytokinesis points to the intriguing possibility of connexin recycling from annular gap junction vesicles to form gap junction plaques as mitosis is completed.