Connexin 43 (Cx43) gap junctions (GJs) electrically couple ventricular cardiomyocytes at the intercalated disc (ID), orchestrating organized organ level contraction with each heartbeat. Disease-related disruption of the Cx43 cytoskeletal trafficking machinery is associated with mislocalization of the Cx43 gap junction protein away from the ID and lethal arrhythmias. We recently found that the majority of intracellular Cx43 cargo is associated with actin, not microtubules, and is either paused or moving slowly. It is not understood why actin is involved in Cx43 trafficking. Using micropatterned HeLa cell pairs and whole-cell automated single particle tracking algorithms, we detected that distinct actin polarity exists in the cell, including highly oriented long fibers associated with fast-moving Cx43 cargo aligned toward actively forming GJ plaques. F-actin disruption with latrunculin A (LatA) leads to a loss of Cx43 cargo directionality toward the cell-cell border, as well as a marked decrease in overall microtubule length. We also found a LatA-dependent biochemical interaction between β-actin and the microtubule plus-end-binding protein EB1, which leads growing microtubules and is a necessary component of the Cx43 forward trafficking machinery. In live cell pairs, F-actin disruption resulted in a decrease in overall EB1 activity and in the number of fully extended microtubules that reach the cell-cell border. Together, these results indicate that actin contributes to the specificity of Cx43 delivery by directing EB1-based microtubule growth toward the cell-cell junction (Please refer to attached diagram).