Although many research have found that colchicine has general therapeutic effect in cardiovascular disease, the therapeutic mechanism in atrial fibrillation has not been clearly studied. To explore whether colchicine plays a role in the treatment of AF by reducing myocardial fibrosis, we performed a series of studies. Rat models of AF were induced by Ach–CaCl₂ to assess the therapeutic effect of colchicine at doses of 0.8 mg/kg on the duration of AF rhythm, degree of myocardial fibrosis, and secretion of inflammatory factors in the serum. RNA-Seq was also performed to elucidate the possible mechanisms by which colchicine might reduce the alleviation of myocardial fibrosis associated with AF. These studies showed that colchicine reduced the duration of AF and the degree of fibrosis in the left atrium and that it significantly reduced the secretion of TGFβ1, activin A, collagen I, and collagen III. These results suggest that colchicine may reduce myocardial fibrosis by (1) inhibiting the TGFβ1/ALK5 and activin A/ALK4 fibrosis pathways; (2) inhibiting the activation, phenotypic transformation, and apoptosis resistance of myocardial fibroblasts; and (3) reducing the synthesis of inflammatory factors and collagen.