Previous studies indicate a mixed lineage kinase, called leucine-zipper and sterile-alpha motif kinase (ZAK) could induce apoptosis through p-JNK activation, and northern blot analysis also reveal that ZAK in heart is the most abundantly level among other organs, implying it might cause cardiac cell death.17β-Estradiol (E2) and Estrogen receptor-alpha (ERα) are researched widely and well known about the function of cardioprotection.Therefore, this study is to clarify the effect of E2/ERα on ZAK-induced cardiac apoptosis in H9c2 cells.We setup three different ZAK tet-on systems (wild type, constitute active and dominant negative) which exhibit different kinase activity to regulate downstream proteins.H9c2 cell apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay, active-caspase-3, 8, 9, p-JNK, and calcineurin levels were evaluated by western blot analysis.In results, significant increases of TUNEL-positive cells and caspase-3, 8, 9 activities after the overexpression of wide type ZAK with tet-on systems were decreased following the treatment of E2/ERα.Inhibitory test (ICI, an inhibitor of E2/ERα) results showed E2/ERα might inhibit apoptosis through regulating p-JNK.According to the above data, we make a conclusion that E2/ERα may have the potential to inhibit ZAK-induced apoptosis in cardiomyoblast H9c2 cells.