Gap junctions (GJ) are specialized cell-cell contacts that provide direct intercellular communication (IC) between eukaryotic cells. Regulation of GJIC by degradation of Cx43 has been a matter of debate over the last two decades and both the proteasome and the lysosome have been implicated. However, the underlying mechanism and molecular players involved remain elusive. In this paper we demonstrate, for the first time, that the ubiquitin ligase Nedd4 is involved in Cx43 ubiquitination. Indeed, depletion of Nedd4 with siRNA resulted in a decrease of the amount of ubiquitin attached to Cx43. Ubiquitinated membrane proteins are often recognized and targeted by endocytic adaptors containing ubiquitin-binding domains, such as Eps15. By coimmunoprecipitation and immunofluorescence we show interaction of Cx43 with Eps15 and colocalization of these proteins mainly at the plasma membrane. Moreover, depletion of Eps15 results in an accumulation of Cx43 at the plasma membrane. Furthermore, the interaction of Eps15 with Cx43 requires the ubiquitin-interacting motif of Eps15 suggesting that the interaction occurs through the ubiquitin attached to Cx43. Data presented in this manuscript are consistent with a new molecular model in which Nedd4-mediated ubiquitination of Cx43 is required to recruit Eps15, through its ubiquitin-interacting motif, and targets ubiquitinated Cx43 to the endocytic pathway. This provides the basis for future studies aiming at identifying the molecular players and mechanisms involved in Cx43 internalization and degradation.