Objectives: This study aimed to investigate the roles of connective tissue growth factor (CTGF) and transforming growth factor-β₁ (TGF-β₁) in atrial fibrosis in patients with chronic atrial fibrillation. Methods: Up to 40 cases involving simple mitral valve replacement surgery were divided into 2 groups: the chronic atrial fibrillation (cAF) group (n = 28) and the sinus rhythm group (n = 12). Echocardiography was used to measure the cardiac cavity size and analyze the cardiac function. Right atrial specimens were obtained during the operation. The collagen volume fraction in the atrial specimens was examined. The mRNA and protein levels of TGF-β₁, Smad3 and CTGF were also investigated. Results: Compared with the sinus rhythm group, the cAF group had higher collagen content in the right atrial tissues. The mRNA and protein levels of TGF-β₁, Smad3 and CTGF were also significantly elevated in the cAF group (p < 0.05). The mRNA and protein levels of TGF-β₁ and CTGF in the cAF group correlated positively with the collagen volume fraction. The positive correlation between the expression of TGF-β₁ and CTGF was also demonstrated. Conclusions: CTGF is upregulated via the TGF-β₁/Smad pathway in the atrial myocardium of cAF patients. Furthermore, the TGF-β₁/Smad pathway may play an important role in the structural remodeling during atrial fibrosis.