Myocarditis is a leading cause of sudden cardiac death in young adults, accounting for 20% of cases under 40 years of age in the United States. Adenovirus is a major etiological agent in this disease yet much remains unknown regarding pathogenesis and predisposition to infection. During adenoviral infection, viral proteins uncouple cell cycle checkpoints for viral DNA genome replication to occur. Connexin43 (Cx43) gap junctions play an essential role in propagation of action potentials to precipitate each heartbeat. Cx43 has also been implicated in cell cycle regulation, leading us to hypothesize that adenovirus targets Cx43 to facilitate viral replication, resulting in arrhythmogenic alterations in cardiac intercellular communication. We find reduced Cx43 expression at the mRNA and protein level within 24 hours of infection with adenovirus type 5 (Ad5) in human epithelial cells. Studies in human induced pluripotent stem cell-derived cardiomyocytes reveal alterations in calcium transient propagation and reduced Cx43 protein levels. Growth factor signaling is activated by early Ad5 proteins and is known to negatively regulate gap junction coupling. We performed genetic screens and have identified the viral protein E4-ORF1 as sufficient in suppression of Cx43 expression and modulation of cardiac transcription factors. siRNA-mediated knockdown of Cx43 protein enhances viral genome replication while transient overexpression of Cx43 significantly inhibits late viral protein synthesis and genome replication. Finally, using dominant-negative trafficking-dead and closed-channel Cx43 mutants, we conclude that inhibition of Ad5 replication by Cx43 is gap junction dependent. Together, these data provide novel insight into molecular mechanisms underlying arrhythmogenic viral myocarditis in addition to understanding the role of Cx43 in adenoviral pathogenesis. Revealing viral mechanisms of gap junction dissolution will contribute not just to the development of novel antiviral therapeutics, but will also provide insight into fundamental Cx43 biology relevant to other forms of heart disease.