Objective To investigate the effects of extracellular signal-regulated kinase 1/2(ERK1/2) pathway inhibitor U0126 on isopreterenol(ISO)-induced atrial fibrosis and connexin 40 (Cx40) remodeling in rats. Methods Thirty-two male SD rats were evenly randomized into control group, DMSO group, ISO (5 mg/\[kg·d\])+DMSO group (fibrosis group), and ISO (5 mg/\[kg·d\])+U0126 (0.5 mg/\[kg·d\])+DMSO group (U0126-treated group ). The corresponding reagents were given to each group once a day and the rats were killed and the myocardial tissues were collected after 7 d. The AngⅡcontents in the myocardial tissues were measured by radioimmunoassay; H-E staining and Masson staining were applied to measure the degree of atrial fibrosis; p-MEK1/2, p-ERK1/2, and Cx40 were detected by immunohistochemistry method. Results (1) The contents of AngⅡ were similar between control group (\[242.133±4.870\] ng/L) and DMSO (\[239.412±1.795\] ng/L) group (P>0.05). Compared with the above two groups, AngⅡ contents in fibrosis group (\[500.250±8.869\] ng/L)and U0126-treated group(\[498.695±9.340\]ng/L) were significantly increased (P all<0.01). (2) Control group and DMSO group had no atrial fibrosis; the degree of atrial fibrosis in U0126-treated group was significantly lower than that in the fibrosis group(P<0.01). (3) p-MEK1/2 and p-ERK1/2 expressions were similar in control group and DMSO group (P>0.05), and those in the fibrosis group were significantly increased compared with control group and DMSO group(P<0.01); the expression in U0126-treated group was similar to those in the control group and DMSO group(P>0.05), and was significantly decreased compared with the fibrosis group(P<0.01). (4) The contents of Cx40 were similar between control group and DMSO group (P>0.05), and Cx40 was distributed in myocardial cell intercalated disc in a linear manner. The content of Cx40 was significantly reduced(P<0.01) in the fibrosis group compared with the control group and DMSO group, with Cx40 distributed in disorder. The content of Cx40 in U0126-treated group was similar to that in the control group and DMSO group(P>0.05)and most of the Cx40 was linearly distributed in myocardial cell intercalated disc. Meanwhile, the reduce degree of Cx40 content in U0126-treated group was significantly decreased than that in the fibrosis group(P<0.01), and some Cx40 was linearly distributed in myocardial cell intercalated disc. Conclusion Long-term Ang Ⅱ elevation in myocardium may be involved in atrial fibrosis and Cx40 remodeling, and U0126 can efficiently improve atrial fibrosis and Cx40 remodeling by inhibiting the activation of ERK1/2 pathway.