Bcl‐2‐associated athanogene3(BAG3) protects the heart and cardiomyocytes from ischaemia/reperfusion (I/R) injury. Although the anti‐apoptosis effect of BAG3 has been demonstrated in multiple cell types, the structural domain of BAG3, which is responsible for its anti‐apoptosis effect, is not well understood. BAG3 protein consists of various characteristic amino acid motifs/regions that permit the interaction of BAG3 with numerous proteins involved in many cellular key pathways. The purpose of this study is to determine whether the proline‐rich (PXXP) domain of BAG3 is necessary for its cellular protection against hypoxia–reoxygenation (H/R) stress by binding to its chaperone, heat shock cognate 71 kDa protein (HSC70). Cell apoptosis induced by H/R was evaluated using propidium iodide (PI) staining, caspase 3/7 activation and TUNEL staining in cultured H9C2 cells. The expression levels of BAG3 and HSC70 were manipulated, where BAG3 or its mutant, which lacked the PXXP domain, was overexpressed using a plasmid and adenovirus vector, and HSC70 expression was silenced using siRNA. Co‐immunoprecipitation (co‐IP) followed by western blot was employed to define the complex of BAG3 binding to its chaperones. The PXXP domain of BAG3 was determined to be critical for BAG3‐mediated attenuation of H9C2 cell apoptosis induced by H/R through the binding of PXXP with HSC70. The abolished cellular protection of BAG3 induced by the knockdown of HSC70 is associated with reduced binding to HSC70. Given that the structural domain PXXP of BAG3 is necessary for the cellular protection of BAG3 from I/R injury, the mechanism revealed in this study indicates that BAG3 may be a therapeutic target in patients undergoing reperfusion after myocardial infarction.