The enhancing effect of synthetic triple-chain amphiphiles on the stability of a vesicle made from phosphatidylcholine was investigated in terms of the leakage of the inner marker. The addition of amphiphiles bearing three hydrophobic chains and two carboxylate groups to the vesicle membrane with a phosphatidylcholine base contributed effectively to strengthening the barrier effect of the membrane of the vesicle. The leakage of entrapped materials was suppressed with increasing ratio of the triple-chain amphiphile in the membrane. To elucidate the factors which bring about the high barrier effect of the triple-chain amphiphile, the zeta potential of the vesicle, microfluidity of the bilayer membrane, pressure-area (pi-A) isotherms of the monolayer membrane, and differential scanning calorimetry of the amphiphiles were studied. Both the increasing surface charge of the vesicle and the enhancement of the hydrophobic interaction near the hydrophile moiety of the bilayer membrane were considered to contribute to the stability of the vesicle containing the triple-chain amphiphile. It was also found that the microfluidity of the bilayer membrane made from triple-chain amphiphiles was less sensitive to temperature change than that of the bilayer membrane made from phosphatidylcholine.