Ischemic heart disease is a leading cause of morbidity and mortality worldwide. Although timely restoration of coronary blood flow (reperfusion) is the most effective therapeutics of myocardial infarction, reperfusion causes further cardiac damage, i.e. ischemia-reperfusion (I/R) injury. β-arrestins (Arrbs) have been traditionally defined as negative regulators of G protein-coupled receptor (GPCR) signalling, but recent studies have shown that they are essential for G protein-independent, GPCR-mediated biased signalling. Several ligands have been reported to be cardioprotective via Arrbs dependent pathway. However, it is unclear whether Arrbs exert receptor-independent physiological or pathological functions in the heart. Here, we sought to determine whether and how Arrbs play a role in regulating cardiomyocyte viability and myocardial remodelling following I/R injury.The expression of β-arrestin 2 (Arrb2), but not β-arrestin 1 (Arrb1), is upregulated in rat hearts subjected to I/R injury, or in cultured neonatal rat cardiomyocytes treated with hypoxia-reoxygenation (H/R) injury. Deficiency of Arrb2 in cultured neonatal rat cardiomyocytes alleviates H/R-induced cardiomyocyte death and Arrb2-/- mice are resistant to myocardial damage caused by I/R injury. In contrast, upregulation of Arrb2 triggers cardiomyocyte death and exaggerates I/R (or H/R)-induced detrimental effects. Mechanically, Arrb2 induces cardiomyocyte death by interacting with the p85 subunit of PI3K, and negatively regulating the formation of p85-PI3K/CaV3 survival complex, thus blocking activation of PI3K-Akt-GSK3β cell survival signalling pathway.We define an upregulation of Arrb2 as a pathogenic factor in cardiac I/R injury, and also reveal a novel GPCR-independent mechanism of Arrb2-mediated cell death signalling in the heart.