Mechanical rigidity of the liposome membrane is often defined by the membrane bending modulus and is one of the determinants of liposome stability, but the quantitative experimental data are still limited to a few kinds of liposomes. Here, we used atomic force microscopy to investigate the membrane bending moduli of liposomes by immobilizing them on bovine serum albumin-coated glass in aqueous medium. The following lipids were used for liposome preparation: egg yolk phosphatidylcholine, dioleoylphosphatidylcholine, hydrogenated soybean phosphatidylcholine, dipalmitoylphosphatidylcholine, 1,2-dioleoyl-3-trimethylammonium-propane, cholesterol, and N-(carbonylmethoxypoly(ethylene glycol) 2000)-1,2-distearoyl-sn-glycero-3-phosphoethanolamine. By using liposomes of various compositions, we showed that the thermodynamic phase state of the membrane rather than the electric potential or liposome surface modification with poly(ethylene glycol) is the predominant determinant of the bending modulus, which decreased in the following order: solid ordered > liquid ordered > liquid disordered. By using the generalized polarization value of the Laurdan fluorescent probe, we investigated membrane rigidity in terms of membrane fluidity. Atomic force microscopic analysis was superior to the Laurdan method, especially in evaluating the membrane rigidity of liposomes containing hydrogenated soybean phosphatidylcholine and cholesterol. Positively charged liposomes with a large bending modulus were taken up by cells more efficiently than those with a small bending modulus. These findings offer a quantitative method of analyzing the membrane rigidity of nanosized liposomes with different lipid compositions and will contribute to the control of liposome stability and cellular uptake efficiency of liposomal formulations intended for clinical use.