Previous studies have demonstrated that 27‐hydroxycholesterol (27‐OHC), a cholesterol metabolite, was involved in the inflammatory process of Alzheimer's disease (AD). The present study aimed to investigate the 27‐OHC‐induced inflammatory damage to neurons and astrocytes and the underlying mechanism(s) accounting for this damage. Human neuroblastoma cells (SH‐SY5Y cells) and rat glioma cells (C6 cells) were treated with vehicle or 27‐OHC (5, 10, or 20 μM) for 24 hr. The levels of secreted interleukin‐1β (IL‐1β), interleukin‐10 (IL‐10), tumor necrosis factor alpha (TNF‐α), and inducible nitric oxide synthase (iNOS) were determined by using an enzyme‐linked immunosorbent assay (ELISA). Immunofluorescence staining was used to determine the cellular expression of toll‐like receptor 4 (TLR4) and transforming growth factor‐β (TGF‐β). The mRNA and protein expression levels of nuclear factor‐κB p65 (NF‐κB p65), nuclear factor‐κB p50 (NF‐κB p50) and cyclooxygenase‐2 (COX‐2) in both SH‐SY5Y and C6 cells were also detected by real‐time PCR and Western blot, respectively. The results of this study showed that 27‐OHC treatment increased secretion of TNF‐α and iNOS and decreased secretion of IL‐10, upregulated expression of TGF‐β, NF‐κB p65 and p50, and downregulated expression of COX‐2 in SH‐SY5Y cells. In C6 cells, treatment with 27‐OHC resulted in decreased secretion of IL‐1β, IL‐10, TNF‐α, and iNOS, and increased expression of TLR4 and TGF‐β. These results suggest that 27‐OHC may cause inflammatory damage to neurons by activating the TGF‐β/NF‐κB signaling pathway and to astrocytes by activating the TLR4/TGF‐β signaling, which results in the subsequent release of inflammatory cytokines.