Gap junctions (GJs), collections of multiple intercellular channels between neighboring cells, are specialized channels facilitating intercellular electrical and chemical communication. GJs are important for synchronizing coupling and coordinated contraction in the heart, and are crucial regulators of heart gene transcription, cardiac development, and protection of ischemic cardiomyocytes through second messenger communication. Identification of proteins that interact with Connexin43 (Cx43), the predominant protein in cardiac GJs, may contribute to the understanding of GJ functional regulation. Using a yeast two-hybrid system, we identified Caveolin-3 (Cav3) as a new Cx43-interacting protein. This interaction was confirmed by co-immunoprecipitation and co-localization experiments. CX43 interacts with Cav3, suggesting that Cav3 may participate in the functional regulation of GJs.