Aim To study the regulatory effect of N-acetylcysteine (NAC) on hypoxia-induced injury of cerebrovascular endothelial cells and its molecular mechanism. Methods Healthy male SD rats were selected, cerebrovascular endothelial cells were isolated and cultured. Cells were divided into normal oxygen group, hypoxia group, 0.5 NAC group(hypoxia+0.5 mol/L NAC), 1.0 NAC group(hypoxia+1.0 mol/L NAC), NAC+8-bAMP group((hypoxia+1.0 mol/L NAC+1.0mol/L 8-bAMP). Cell proliferation activity was detected by MTS assay, apoptotic rate was detected by TUNEL assay, oxidative stress index was detected by kit, apoptotic gene and AMPK/SIRT1 pathway molecule expression was detected by western blot. Results OD₄₉₀ value, T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in hypoxia group were significantly lower than those in normoxia group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of hypoxia group were significantly higher than those in normoxia group. OD₄₉₀ value, content of T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in 0.5 NAC group, 1.0 NAC group were significantly higher than those in hypoxia group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of 0.5 NAC group, 1.0 NAC group were significantly higher than those in hypoxia group. OD₄₉₀ value, content of T-AOC and expression of Bcl-2, p-AMPK, SIRT1 in NAC+8-bAMP group were significantly lower than those in 1.0 NAC group, while apoptotic rate, contents of ROS, MDA, 8-OHDG and expression of Caspase-3, Cyt-C, Bax of NAC+8-bAMP group were significantly higher than those in 1.0 NAC group. Conclusion NAC can alleviate oxidative stress and mitochondrial apoptosis-mediated injury of cerebrovascular endothelial cells by activating AMPK/SIRT1 pathway.