Zing finger protein 580 (ZFP580) is a novel Cys2-His2 zinc-finger transcription factor that has an anti-apoptotic role in myocardial cells. It is involved in the endothelial transforming growth factor-beta 1 (TGF-beta 1) signal transduction pathway as a mothers against decapentaplegic homolog (Smad) 2 binding partner. The aim of the present study was to determine the involvement of ZFP580 in TGF-beta 1-mediated cytoprotection against chemical hypoxia-induced apoptosis, using H9c2 cardiac myocytes. Hypoxia was chemically induced in H9c2 myocardial cells by exposure to cobalt chloride (CoCl2). In response to hypoxia, cell viability was decreased, whereas the expression levels of hypoxia inducible factor-1 alpha and ZFP580 were increased. Pretreatment with TGF-beta 1 attenuated CoCl2-induced cell apoptosis and upregulated ZFP580 protein expression; however, these effects could be suppressed by SB431542, an inhibitor of TGF-beta type I receptor and Smad2/3 phosphorylation. Furthermore, suppression of ZFP580 expression by RNA interference reduced the anti-apoptotic effects of TGF-beta 1 and thus increased CoCl2-induced apoptosis. B-cell lymphoma (Bcl)-2-associated X protein/Bcl-2 ratio, reactive oxygen species generation and caspase-3 activation were also increased following ZFP580 inactivation. In conclusion, these results indicate that ZFP580 is a component of the TGF-beta 1/Smad signaling pathway, and is involved in the protective effects of TGF-beta 1 against chemical hypoxia-induced cell apoptosis, through inhibition of the mitochondrial apoptotic pathway.