Atrial fibrillation (AF) is the most common form of sustained arrhythmia, its incidence increases with age and it is associated with extensive atrial remodelling. The mitogen activated protein kinase cascade (MAPK) is implicated in regulating the pathogenesis of AF. MAPKs regulate fibrosis in the ventricle in aged/hypertrophied hearts. Fibrosis is associated with cardiac arrhythmias, including AF. To establish the role of the MAPK pathway in atrial function in ageing, we have developed an atrial specific conditional knockout (ACKO) mouse model where a central component of this pathway; MKK4, has been specifically deleted from the atria using the Cre-Loxp system. To determine effect of MKK4 deficiency on atrial electrical function, surface ECG was performed on MKK4f/f and MKK4ACKO mice anesthetized with isoflurane (2.5%) and the recordings were examined. With age, MKK4 atrial-knock outmice (MKK4ACKO) became more susceptible to atrial arrhythmias with characteristic slow atrial conduction comparing with MKK4F/F mice. In parallel, an increased interstitial fibrosis, up-regulated TGF-β1 signalling and dysregulation of matrix metalloproteinases and their inhibitory enzymes were observed in the atrium of MKK4ACKO mice in contrast to control MKK4F/F mice. Knockdown MKK4 in in-vitro cultured cardiomyocytes led to a more sensitivity to Ang-II stimulation induced TGF-β1 signaling activation, such change in turn enhances paraocrine expression of profibrotic molecules in cultured cardio-fibroblasts, suggesting a cross-talk between two cell types on profibrotic signaling. Further study in AF atrial tissue in human revealed an association of down-regulation of MKK4 and activation profibrotic signalling including a significant increase in expression of col1a1, col3a1, ctgf and downregulation of mmp2. Taken together, our results for the first time have established a critical role of MKK4 in age-related atrial structural remodeling associated with AF. As a negative regulator of TGF-β1 profibrotic signaling pathway, MKK4 is therefore likely to be a new molecular therapeutic target for developing anti-AF drugs.