AbstractGap junction (GJ) intercellular communication (GJIC) is vital to ensure proper cell and tissue function. GJ are multimeric structures composed of proteins called connexins. Modifications on stability or subcellular distribution of connexins have a direct impact on the extent of GJIC. In this study we have investigated the role of the proteasome in regulation of connexin 43 (Cx43) internalization. Although the participation of both the proteasome and lysosome has long been suggested in Cx43 degradation, the molecular mechanisms whereby proteasome contributes to regulate Cx43 internalization and intercellular communication are still unclear. The results presented in this study envision a new mechanism whereby proteasome regulates GJIC by modulating interaction between Cx43 and ZO‐1. Immunoprecipitation experiments, in the presence of proteasome inhibitors, together with immunofluorescence data indicate that the proteasome regulates interaction between Cx43 and ZO‐1. Overexpression of the PDZ2 domain of ZO‐1 and the expression of Cx‐43 fused in frame with a V5/HIS tag, suggest that interaction between the two proteins occurs through the PDZ2 domain of ZO‐1 and the C‐terminus of Cx43. When interaction between Cx43 and ZO‐1 is reduced, as in the presence of proteasome inhibitors, Cx43 accumulates, forming large GJ plaques at plasma membrane. Data presented in this article suggest a new pathway whereby alterations in proteasome activity may impact on GJIC as well as on non‐junctional communication with extracellular environment, contributing to cell and tissue dysfunction. J. Cell. Biochem. 102: 719–728, 2007. © 2007 Wiley‐Liss, Inc.