Purpose : Glaucoma is a severe and irreversible ocular disease characterized by progressive optic neuropathy with the loss of retinal ganglion cells (RGC). One of the established causes of RGC apoptosis is Müller Glial Cell (MGC) activation, called “retinal gliosis”. MGC are the most numerous glial cells in the retina and one of their roles is to maintain cholesterol homeostasis. A lack or an excess of cholesterol in neurons can cause neuro-degeneration. Cholesterol 24S-hydroxylase (CYP46A1) is present in RGC where it catalyses the formation of 24S-hydroxycholesterol (24S-OHC), and thereby participates to cholesterol elimination. CYP46A1 is overexpressed during glaucoma, suggesting that 24S-OHC would be a potent inducer of retinal gliosis. We hypothesize that 24S-OHC homeostasis impairs MGC membrane dynamics involving lipid rafts and as a consequence induces retinal gliosis. Methods : MGC were grown in vitro from retinas of young rats. They were treated with 10µM 24S-OHC or ethanol for 2 min or 6 h. Lipid-rafts of MGC membranes were obtained after lysis with 1% Lubrol and 20 h of ultracentrifugation at 180,000 g. Western-blotting of GFAP, Vimentin, Nestin, Connexin43, Kir4.1, Aquaporine 1, Crystalline alpha B, phosphorylated and non-phosphorylated p38 MAPK and p42-44 MAPK was done for each sample. Ganglioside GM3 (monosialodihexosylganglioside), a marker of lipid rafts, in the MGC membrane in raft and non-raft fractions was quantitated by liquid-chromatography-mass spectrometry (LC-MS). MGC membrane fluidity was evaluated by fluorescence spectrospcopy using an anisotropy probe (TMA-DPH). Results : In vitro treatment of MGC with 24S-OHC tended to increase vimentin expression and p38 and p42-44 MAPK. No difference on GFAP expression was observed. Following the treatment, connexin43 tended to be increased and localized in the raft fractions. LC-MS analysis showed that the treatment increased the proportion of GM3 in the raft fractions. Treatment with 24S-OHC also increased MGC membrane anisotropy. Conclusions : The over-expression of 24S-OHC by RGC during glaucoma may disturb lipid raft organization and trigger different cellular signaling pathways including p42-44 and p38 MAPK which may contribute to retinal gliosis.