Therefore, we focused on the changes in cultured neurons in subsequent experiments. We revealed that the number of 5mC-positive neurons were increased until 1 h after start of N-methyl-D-aspartate (NMDA) treatment and then decreased to the control level. Next, we examined whether DNA methyltransferase (DNMT) inhibitors were able to protect neurons against NMDA treatment. NMDA-treated neurons were protected by different types of DNMT inhibitors, zebularine and RG108 that were accompanied by inhibition of DNA methylation. These results indicated that DNA methylation after ischemic injury may have a role for initiator of neuronal cell death.