Differentiation and activation of fibroblasts into myofibroblasts which express alpha-smooth muscle actin (alpha-SMA) are essential for wound healing and tissue repair. Change in fibroblast properties is initiated by transforming growth factor beta (TGF-beta). Here, we sought to investigate whether connexin43 (Cx43), a gap-junctional protein, contributes to differentiation of cardiac fibroblasts to myofibroblasts. In cultured neonatal rat cardiac fibroblasts, we found that expression of alpha-SMA increases in parallel with Cx43 by using immunocytochemistry, and that knockdown of the endogenous Cx43 activity with antisense oligodeoxynucleotides (AS) inhibits alpha-SMA expression significantly, while overexpression of Cx43 increases alpha-SMA expression remarkably. These findings demonstrate that Cx43 contributes to TGF-beta signaling to regulate alpha-SMA expression. Thus, we propose a novel physiologic function of Cx43, which plays a critical role in the pathological activation of cardiac fibroblasts in the myocardial fibrosis associated with heart failure.