Activated microglia are associated with deposits of aggregated proteins within the brains of patients with Alzheimer’s disease (AD), Parkinson’s disease (PD) and prion diseases. Since the cytokines secreted from activated microglia are thought to contribute to the pathogenesis of these neurodegenerative diseases, compounds that suppress cytokine production have been identified as potential therapeutic targets. CD14 is a glycosylphosphatidylinositol (GPI)- anchored protein that is part of a receptor complex that mediates microglial responses to peptides that accumulate in prion disease (PrP82-146), AD (amyloid-β (Aβ)42) and PD (α-synuclein (αSN)). As some GPI-anchored proteins are released from cells by treatment with glimepiride, a sulphonylurea used for the treatment of diabetes, the effects of glimepiride upon CD14 expression and cytokine production from cultured macrophages were studied. RAW 264 cells and microglial cells were treated with glimepiride or phosphatidylinositol (PI)-phospholipase C (PLC) and the expression of cell receptors was analysed by ELISA and immunoblot. Treated cells were subsequently incubated with Aβ42, αSN, PrP82-146 or lipopolysaccharide (LPS) and the amounts of Toll-like receptor (TLR)-4, tumour necrosis factor (TNF), interleukin (IL)-1 and IL-6 measured. Glimepiride released CD14 from RAW 264 cells and microglial cells. Pre-treatment with glimepiride significantly reduced TNF, IL-1 and IL-6 secretion from RAW 264 and microglial cells incubated with LPS, Aβ42, αSN and PrP82-146. Glimepiride also reduced the LPS, Aβ42, αSN and PrP82-146-induced translocation of TLR-4 into membrane rafts that is associated with cell activation. These effects of glimepiride were also seen after digestion of RAW 264 cells with PI-phospholipase C (PLC). In addition, the effects of glimepiride were blocked by pharmacological inhibition of GPI-PLC. The cytokine production was CD14-dependent; it was reduced in microglia from CD14 knockout mice and was blocked by antiserum to CD14. RAW 264 and microglial cell responses to Aβ1–42, αSN, PrP82-146 and LPS are dependent upon CD14 expression. Glimepiride induced the shedding of CD14 from cells by activation of GPI-PLC and consequently reduced cytokine production in response to Aβ42, αSN, PrP82-146 and LPS. These results suggest that glimepiride acts as a novel anti-inflammatory agent that could modify the progression of neurodegenerative diseases.