BACKGROUND: Pathological cardiac hypertrophy can result in heart failure. Protein dephosphorylation plays a primary role in the mediation of various cellular processes in cardiomyocytes. Here, we investigated the effects of a protein tyrosine phosphatase, PRL2 (phosphatase of regenerative liver 2), on pathological cardiac hypertrophy. METHODS: The PRL2 knockout mice were subjected to angiotensin II infusion or transverse aortic constriction to induce myocardial hypertrophy and cardiac dysfunction. RNA-sequencing analysis was performed to explore the underlying mechanisms. Mass spectrometry and bio-layer interferometry assays were used to identify AMPKα2 (AMP-activated protein kinase α2) as an interacting protein of PRL2. Mutant plasmids of AMPKα2 were used to clarify how PRL2 interacts and dephosphorylates AMPKα2. RESULTS: A significant upregulation of PRL2 was observed in hypertrophic myocardium tissues in mice and patients with heart failure. PRL2 deficiency alleviated cardiac hypertrophy, fibrosis, and dysfunction in mice challenged with angiotensin II infusion or transverse aortic constriction. Transcriptomic and biochemical analyses showed that PRL2 knockout or silence maintained AMPK CONCLUSIONS: This study reveals that PRL2, as a novel AMPK-regulating phosphatase, promotes mitochondrial instability and hypertrophic injury in cardiomyocytes and provides PLR2 as a potential target for future drug development treating heart failure.