Oleuropein, glycosylated secoiridoid present in the olive leaves, is known as an important antioxidant phenolic compound. We studied the antioxidant effect of low dose of oleuropein aglycone (3,4-DHPEA-EA) and oleuropein aglycone peracetylated (3,4-DHPEA-EA(P)) in murine C2C12 myocytes treated with hydrogen peroxide (H2O2). Both compounds were used at a concentration of 10 μM and were able to inhibit cell death induced by H2O2 treatment, with 3,4-DHPEA-EA(P) being more. Under our experimental conditions H2O2 efficiently induced the phosphorylated-active form of JNK and of its downstream target c-Jun. We demonstrated, by Western blot analysis, that 3,4-DHPEA-EA(P) was efficient in inhibiting the phospho-active form of JNK. This data suggest that growth arrest and cell dead of C2C12 proceeds via the JNK/c-Jun pathway. Moreover, we demonstrated that 3,4-DHPEA-EA(P) affects myogenesis of C2C12 cells; because the MyoD mRNA levels and the differentiation process are restored after treatment with 3,4-DHPEA-EA(P). Overall, the results indicate that 3,4-DHPEA-EA(P) prevents ROS-mediated degenerative process by functioning as an efficient antioxidant.