Human erythrocytes contain abundant cholesterol as membrane lipids. Cholesterol contributes to the stability and function of the membrane. Membrane stability of the erythrocyte has been mainly examined under hypotonic conditions, but not under high hydrostatic pressure. So, the effect of cholesterol on the membrane stability of human erythrocyte was examined under a pressure of 200 MPa. As with hypotonic hemolysis, the pressure-induced hemolysis was enhanced by depletion of cholesterol from the intact erythrocyte membrane, whereas suppressed by cholesterol loading to the intact one. Enhancement of such hemolysis was associated with the suppression of fragmentation, whereas the hemolysis was suppressed by the facilitation of vesiculation. Cholesterol induced the tight linkage of the lipid bilayer with cytoskeleton. Taken together, these results suggest that the erythrocyte membrane stability is affected by such tight linkage by cholesterol.