Recent studies showed that stroke extensively alters cerebral microRNA (miRNA) expression profiles and several miRNAs play a role in mediating ischemic pathophysiology. We currently evaluated the significance of miR-29c, a highly expressed miRNA in rodent brain that was significantly down-regulated after focal ischemia in adult rats as well as after oxygen-glucose deprivation in PC12 cells. Bioinformatics indicated that DNA methyltransferase 3a (DNMT3a) is a major target of miR-29c and co-transfection with premiR-29c prevented DNMT3a 3'UTR vector expression. In PC12 cells, treatment with premiR-29c prevented OGD-induced cell death (by 58 ± 6%; p<0.05). Furthermore, treatment with antagomiR-29c resulted in a 46 ± 5% cell death in PC12 cells. When rats were treated with premiR-29c and subjected to transient focal ischemia, post-ischemic miR-29c levels were restored and the infarct volume decreased significantly (by 34 ± 6%; p<0.05) compared to control premiR treated group. DNMT3a siRNA treatment also significantly curtailed the post-OGD cell death in PC12 cells (by 54 ± 6%; p<0.05) and decreased the post-ischemic infarct volume in rats (by 30 ± 5%; p<0.05) compared to respective control siRNA treated groups. The miR-29c gene promoter showed specific binding sites for the transcription factor REST and the miR-29c promoter vector expression was curtailed when cotransfected with a REST expressing plasmid. Furthermore, treatment with REST siRNA prevented the post-ischemic miR-29c down-regulation and DNMT3a induction in PC12 cells and curtailed ischemic cell death (by 64 ± 9%; p<0.05) compared to control siRNA treatment. These studies suggest that miR-29c is a pro-survival miRNA and its down-regulation is a promoter of ischemic brain damage by acting through its target DNMT3a. Furthermore, REST is an upstream transcriptional controller of miR-29c and curtailing REST induction prevents miR-29c down-regulation and ischemic neuronal death.