We demonstrated for the first time in a canine model of AF that prolonged rapid atrial pacing increased exosome release and that the blockade of exosome release with GW4869 suppressed AF by alleviating atrial fibrosis, which was related to pro-fibrotic miR-21-5p enriched in exosomes and its downstream TIMP3/TGF-β1 signaling pathway. Here, TIMP3 is linked to atrial fibrillation.