Purpose The catabolism of cholesterol in neurons leads to a more hydrophilic compound soluble form, the 24‐S‐hydroxycholesterol by means of an enzyme the CYP46A1.The aim of this study was to analyse the implication of 24‐S‐hydroxycholesterol (24‐S‐OHC) on Müller glial cells (MGC) membrane dynamics in the rat. Methods MGC were grown in vitro from retinas of 10‐day‐old Long Evans rats. Cells were treated with 24‐S‐OHC (treatment) or ethanol (control) for 2 min or 6 h. From twenty millions of MGC in each group, lipid‐rafts were obtained after a 1% Lubrol lysis and an ultra centrifugation (180,000 g – 20 h – 4°C). The following proteins: caveolin, flottilin, connexin 30 and 43, CRALBP, DHAPAT, GFAP and vimentin were analysed using Western blotting on all fractions (lipid‐rafts and non‐rafts). MGC membrane fluidity was studied in vitro with two different techniques: anisotropy measurements performed with the lipophilic fluorescent probe TMA‐DPH and fluorescence recovery after photobleaching (FRAP) observed using confocal microscopy. Results 24‐S‐OHC treatment on in vitro MGC increased the expression of GFAP and delocalized GFAP in the lipid‐raft fraction; 24‐S‐OHC treatment induced a delocalization of DHAPAT protein out of the lipid‐rafts fraction. Anisotropy was decreased with the 24‐S‐OHC treatment (difference: 5.1 × 10−3; p < 0.01) revealing an increase of the membrane fluidity. This increase was confirmed by the FRAP technique, which showed a shorter time of fluorescence recovery for the treated cells. Conclusions This study showed that 24‐S‐OHC could be a candidate leading a key role in the activation of MGC, disturbing lipid‐raft organization by changing the localization of signalization proteins and increasing membrane's fluidity.