Globotriaosylceramide (Gb3 ) is a glycosphingolipid present in the plasma membrane that is the natural receptor of the bacterial Shiga toxin. The unsaturation level of Gb3 acyl chains has a drastic impact on lipid bilayer properties and phase behaviour, and on many Gb3-related cellular processes. For example: the Shiga toxin B subunit forms tubular invaginations in the presence of Gb3 with an unsaturated acyl chain (U-Gb3 ) while in the presence of Gb3 with a saturated acyl chain (S-Gb3 ) such invagination does not occur. We have used all-atom molecular dynamics simulations to investigate the effects of the Gb3 concentration and its acyl chain saturation on the phase behaviour of a mixed bilayer of dioleoylphosphatidylcholine and Gb3 . The simulation results show that: 1) The Gb3 acyl chains (longer tails) from one leaflet interdigitate into the opposing leaflet and lead to significant bilayer rigidification and immobilisation of the lipid tails. S-Gb3 can form a highly ordered, relatively immobile phase which is resistant to bending while these changes for U-Gb3 are not significant. 2) At low concentrations of Gb3 , U-Gb3 and S-Gb3 have a similar impact on the bilayer reminiscent of the effect of sphingomyelin lipids and 3) At higher Gb3 concentrations, U-Gb3 mixes better with dioleoylphosphatidylcholine than S-Gb3 . Our simulations also provide the first molecular level structural model of Gb3 in membranes.