Ischemic stroke causes cerebral hypoxia, while long non-coding RNA regulator of reprogramming is associated with hypoxia. To find a new intervention target to protect hypoxic cerebral tissue and a potential biomarker from reflecting the severity of hypoxia after ischemic stroke, our research explored the expression pattern and function of the regulator of reprogramming in cerebral hypoxia-induced injury. The expression pattern and the function of the regulator of reprogramming were explored in mice with middle cerebral artery occlusion, and human brain microvascular endothelial cells underwent oxygen-glucose deprivation treatment. A case-control study, including 223 ischemic stroke patients and 155 controls were also conducted to investigate its correlation with ischemic stroke clinical characteristics. Results showed that the regulator of reprogramming increased significantly in middle cerebral artery occlusion in mice (P < 0.05), and its level remained stable within 2 to 48 h after the implement of middle cerebral artery occlusion. Oxygen-glucose deprivation up-regulated the expression of regulator of reprogramming, and regulator of reprogramming promoted ASK-1/STRAP/14-3-3 complex formation to inhibit the activation of TNF-α/ASK-1-mediated apoptosis of human brain microvascular endothelial cells, while small interfering ribonucleic acid (RNA) targeting regulator of reprogramming amplified these effects. Regulator of reprogramming increased and maintained stable within 3 to 48 h of ischemic stroke onset in patients, and was negatively associated with the National Institutes of Health Stroke Scale (NIHSS) (r = -0.708, P < 0.001), high sensitivity C-reactive protein (Hs-CRP) (r = -0.683, P < 0.001) level, infarct volume (r = -0.579, P < 0.001), and modified Rankin scale (mRS) (r = -0.728, P < 0.001). These results indicate that the regulator of reprogramming can alleviate cerebral hypoxia-induced injury by suppressing TNFα-induced apoptosis of vascular endothelial cells.