Ischemic preconditioning is an endogenous protective mechanism that may be triggered by exposure to hydrogen peroxide (H2O2). However, the exact mechanisms underlying preconditioning remain to be fully understood. Ataxia-telangiectasia mutated (ATM) is regarded as an essential endogenous protective protein against stress. The aim of the present study was therefore to investigate whether ATM mediates H2O2 preconditioning. Preconditioning of Neuro‑2a (N2a) cells with 100 µM H2O2 for 90 min resulted in protection from injury induced by a long period of exposure to 600 µM H2O2. In addition, preconditioning with 100 µM H2O2 activated ATM and increased ATM mRNA and protein expression levels in N2a cells. Furthermore, the protective effects induced by H2O2 preconditioning were attenuated by pretreatment with the ATM inhibitor, Ku55933, or ATM small interfering RNA. In conclusion, these findings suggested that ATM is involved in H2O2 preconditioning‑mediated protection against oxidative stress‑induced injury in N2a cells. To the best of our knowledge, the present study demonstrated, for the first time, that the ATM protein is a key mediator of H2O2 preconditioning.