Rationale: Delivery of connexin 43 (Cx43) to the intercalated disc is a continuous and rapid process critical for intercellular coupling. By a pathway of targeted delivery involving microtubules and actin cytoskeleton rest stops, Cx43 hemichannels are efficiently trafficked to adherens junctions at intercalated discs. It has recently been shown that an internally translated isoform of Cx43, GJA1-20k, facilitates full-length Cx43 trafficking in cell lines, although the mechanism remains unknown. Objective: We explored the mechanism by which GJA1-20k regulates the trafficking of full-length Cx43 to intercalated discs. Methods and Results: In vivo overexpression of exogenous GJA1-20k, administered via AAV9-mediated gene delivery, increases the delivery of full length Cx43 to intercalated discs in mouse hearts. Using electron microscopy and fluorescence microscopy, together with biochemical co-immunoprecipitation, we found in micro-patterned HeLa cells and cardiomyocytes that GJA1-20k not only substantially increases the number and length of actin fibers, but can also rescue the effect of actin disruption. GJA1-20k complexes with actin and tubulin, improving microtubule targeting to cell-cell borders in the setting of actin disruption. Actin is also disrupted in acute ischemia-reperfusion (IR) injury. The ex vivo rescue potential of GJA1-20k was further tested in mouse hearts subjected to myocardial IR injury. As compared to control GFP and full length GAJ1-43k, only GJA1-20k gene transfer significantly improves the targeting of Cx43 to intercalated discs following IR injury. Conclusions: These results indicate that GJA1-20k positively modulates actin cytoskeleton to facilitate microtubule-based Cx43 trafficking machinery, promoting the delivery of full-length Cx43 to cardiac cell-cell junctions under normal and ischemic conditions. Therefore, up regulation of GJA1-20k is a potential therapeutic option to reverse the loss of Cx43 in IR injuries.