Irisin is a recently identified myokine which brings increases in energy expenditure and contributes to the beneficial effects of exercise through the browning of white adipose tissues. However, its effects in the heart remains unknown. This study sought to determine the effects of irisin on hypoxia/reoxygenation injury and its relationship with HDAC4. Wild type and stable HDAC4-overexpression cells were generated from H9c2 cardiomyoblasts. HDAC4 overexpression cells and wild type H9c2 cells were exposed to 24 hours of hypoxia followed by one hour of reoxygenation in vitro in the presence or absence of irisin (5 ng/ml). Cell cytotoxicity, apoptosis, mitochondrial respiration, and mitochondrial permeability transition pore (mPTP) were determined. Western blotting was employed to determine active-caspase 3, annexin V, and HDAC4 expression. As compared to wild type H9c2 group, HDAC4 overexpression remarkably led to a great increase in cell death as evident by the increased lactate dehydrogenase (LDH) leakage, ratio of caspase-3-positive cells as well as the upregulated levels of active-caspase 3 and annexin V shown by western blot analysis. In addition, HDAC4 overexpression also induced much severe mitochondrial dysfunction, as indicated by apoptotic mitochondria and increased mPTP. However, irisin treatment significantly attenuated all of these effects. Though irisin treatment did not influence the expression of HDAC4 at the transcriptional level, western blot analysis showed that HDAC4 protein levels decreased in a time-dependent way after administration of irisin, which is associated with the degradation of HDAC4 mediated by small ubiquitin-like modification (SUMO). Our results are the first to demonstrate that the protective effects of irisin in cardiomyoblasts exposed to hypoxia/reoxygenation might be associated with HDAC4 degradation.