The disturbance in cholesterol metabolism has been considered as a cause of alzheimer's disease (AD), which dues to the oxidative damage and cell apoptosis in the brain. We aimed to investigate the toxicity and mechanism of AD-like pathology caused by cholesterol oxidation metabolite 27-hydroxycholesterol (27-OHC) in astrocyte cells. C6 cells were treated with 0, 5, 10, 20 µM 27-OHC for 24 h (h). The cell viability was monitored by using methyl thiazolyl tetrazolium test, generation of reactive oxygen species (ROS) was measured by using 2', 7'-dichlorodihydrofluorescein diacetate fluorescent probe under flow cytometry. The concentrations of 8-hydroxyl deoxyguanosine, the anti-oxidative enzymes such as total superoxide dismutase (tSOD), reduced glutathione (rGSH) and glutathione peroxidase (GSH-Px) were tested by using enzyme-linked immunosorbent assay and enzymic method, respectively. The gene and protein expression of nuclear factor E2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), NAD(P)H dehydrogenase quinone 1 (NQO1) and γ-glutamylcysteine synthetase (γ-GCS) in C6 cells were detected by quantitative western blot analysis and real-time PCR analysis. Moreover, the Nrf2 expressions in both of the cytoplasm and nucleus were detected with western blot analysis, and the localization of Nrf2 was performed by immunocytochemistry and confocal microscopy. 27-OHC increased the levels of ROS and decreased the levels of tSOD, rGSH, GSH-Px in C6 cells dose-dependently. In addition, 27-OHC down regulated the expressions of Nrf2, HO-1, NQO1 and γ-GCS at both of gene and protein levels, while Nrf2 expression in the cytoplasm showed decreased trend after incubated for 24 h with 27-OHC. The cholesterol metabolite 27-OHC is toxic to C6 cells and contributed to oxidative damage via regulating the Nrf2 signaling pathway. Our results suggest that 27-OHC may represent a common pathogenic factor in AD.