--- - |2+ Abstract Aim Atrial fibrillation (AF) is an important cause of morbidity and mortality in the modern world. Loss‐of‐function mutation in the zinc finger homeobox 3 gene (ZFHX3) is associated with increased risk of AF. MicroRNAs (miRNAs) participate in arrhythmogenesis, and thus miRNA modulators may be applicable as therapeutic modalities for AF. However, the altered miRNA profiles after ZFHX3 knockdown (KD) remain unclear. This study aimed to analyse the changes of miRNA expression in loss‐of‐function of ZFHX3 and the effect of miRNA modulation on atrial arrhythmias in this model. Methods We performed small RNA deep sequencing on ZFHX3‐KD and control HL‐1 mouse atrial myocytes. The effect of miRNAs on ZFHX3‐dependent atrial arrhythmia was evaluated through in vitro and in vivo assays in mice. Results Among the differentially expressed miRNAs, 11 were down‐regulated and 6 were up‐regulated after ZFHX3 KD. Quantitative real‐time PCR analysis confirmed that after ZFHX3 KD, miR‐133a and miR‐133b were significantly down‐regulated, whereas miR‐184 was the most significantly up‐regulated. DIANA‐miRPath analysis suggested that miR‐133a/b down‐regulation increases the targeted signalling of miR‐133 (ie, adrenergic, Wnt/calcium and fibroblast growth factor receptor 1 signalling), which could contribute to pathological remodelling of cardiomyocytes. These results were confirmed through Western blotting. After transfection of miR‐133a/b mimics in ZFHX3‐KD cells, miR‐133a/b levels increased, accompanied by the inhibition of their target signalling. Treatment with miR‐133a/b mimics diminished ZFHX3 KD–induced atrial ectopy in mice. Conclusion ZFHX3‐KD promotes distinct miRNA expressional changes in atrial myocytes. MiR‐133a/b mimics may reverse signalling of ZFHX3 KD‐mediated cardiac remodelling and atrial arrhythmia. - 'Acta Physiologica, Volume 227, Issue 3, November 2019. '