Estrogens can regulate apoptosis in various cellular systems. The present study shows that 17β-estradiol (E₂), at physiological concentrations, abrogates DNA damage, poly (ADP-ribose) polymerase cleavage, and mitochondrial cytochrome c release induced by H₂O₂ or etoposide in mouse skeletal muscle C2C12 cells. This protective action, which involved PI3K/Akt activation and Bcl-2 associated death agonist (BAD) phosphorylation, was inhibited by antibodies against the estrogen receptor (ER) α or β isoforms, or transfecting siRNA specific for each isoform. The inhibition of the antiapoptotic action of E₂ at the mitochondrial level was more pronounced when ER-β was immunoneutralized or suppressed by mRNA silencing, whereas transfection of C2C12 cells with either ER-α siRNA or ER-β siRNA blocked the activation of Akt by E₂, suggesting differential involvement of ER isoforms depending on the step of the apoptotic/survival pathway evaluated. These results indicate that E₂ exerts antiapoptotic effects in skeletal muscle cells which are mediated by ER-β and ER-α and involve the PI3K/Akt pathway.