AbstractAmine oxidase copper‐containing 3 (AOC3) is a member of the semicarbazide‐sensitive amine oxidase enzyme family. It acts as an ectoenzyme catalysing the oxidative deamination of primary amines and generating hydrogen peroxide (H2O2). While AOC3 is implicated in cardiovascular diseases such as atherosclerosis, its role in cardiac remodelling after myocardial infarction (MI) is unclear. In this study, we first confirmed a long‐term upregulation of AOC3 in both cardiac myofibroblasts after MI in vivo and angiotensin II (ANGII)‐treated cardiac fibroblasts in vitro. AOC3 knockdown not only inhibited the activation of cardiac fibroblasts induced by ANGII but also alleviated cardiac fibrosis in mice after MI. Using sh‐AOC3 lentiviruses, exogenous recombinant AOC3 (r‐AOC3), semicarbazide (an AOC3 inhibitor), and catalase (a hydrogen peroxide scavenger) treatments, we also demonstrated that AOC3 promoted H2O2 generation, increased oxidative stress, and enhanced ERK1/2 activation, which were responsible for the activation of cardiac fibroblasts. In particular, AOC3 knockdown also improved cardiac function and hypertrophy after MI. Through a coculture system, we confirmed that AOC3 expressed on cardiac myofibroblasts was able to enhance oxidative stress and induce hypertrophy of cardiomyocytes by promoting H2O2 generation. Similarly, r‐AOC3 promoted H2O2 generation and resulted in oxidative stress and hypertrophy of cardiomyocytes, which were almost inhibited by both semicarbazide and catalase. In conclusion, AOC3 plays a critical role in cardiac fibrosis and hypertrophy after MI by promoting the generation of H2O2. AOC3 is a promising therapeutic target against cardiac remodelling. © 2023 The Pathological Society of Great Britain and Ireland.