Anchored Protein Kinase-A (PKA) bound to A-Kinase Anchoring Protein (AKAP) mediates the effects of localized increases in cAMP in defined subcellular microdomains and retains the specificity in cAMP–PKA signaling to distinct extracellular stimuli. Gap junctions are pores between adjacent cells constituted by connexin (Cxs) proteins that provide a means of communication, electrical impulse propagation, and transfer of small molecules. One of the most widely and highly expressed Cxs is Cx43, which was shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane-associated protein that can serve as an AKAP and hold an anchored pool of PKA. We found PKA to be part of a macromolecular signaling complex with ezrin and gap junction protein Cx43 that provides cAMP-mediated control of gap junction communication. We established that an inhibition of PKA activity or disruption of the ezrin–Cx43 interaction abolished PKA-dependent phosphorylation of Cx43 as well as gap junction communication. In vitro studies using peptide arrays, together with mass spectrometry, pointed to serine 369 and 373 of Cx43 as the major PKA phosphorylation sites. A combination of knockdown and reconstitution experiments and gap-FLIP assays with mutant Cx43 containing single or double phosphoserine–mimicking amino acid substitutions in putative PKA phosphorylation sites demonstrated that phosphorylation of S369 and S373 mediated gap junction communication. We propose that the PKA–ezrin–Cx43 macromolecular complex regulating gap junction communication constitutes a general mechanism to control opening of Cx43 gap junctions by phosphorylation in response to cAMP signaling in various cell types (i.e., heart, liver, and placenta).