Sphingoid base C4 hydroxylation is required for syringomycin E action on the yeast plasma membrane. Detergent‐insoluble glycolipid‐enriched membranes (DIGs) from a yeast strain lacking C4 hydroxylated sphingoid bases (sur2 Δ) are composed of linear membrane fragments instead of vesicular structures observed for wild‐type DIGs, though they have similar lipid compositions and amounts of DIG marker proteins. Light‐scattering bands collected from sur2 Δ after centrifugation of Triton X‐100‐treated cell lysates in continuous density gradients have lower buoyant densities than that of the wild‐type. The results show that C4 hydroxylation influences the physical and structural properties of DIGs and suggest that syringomycin E interacts with lipid rafts.