This study guided the potential of composite phospholipid-chitosan vesicles (chitosomes) as novel delivery systems for four kinds of carotenoids, lycopene, β-carotene, lutein and canthaxanthin. Chitosomes were successfully achieved by combing the liposomal preparation and the layer self-assembly deposition technique. The physical properties of such colloidal nanocarriers were characterized by microstructure, particle size and distribution using transmission electron microscopy and dynamic light scattering. It was demonstrated that chitosan can flatly adsorb onto the membrane surface through electrostatic attraction, inducing charge inversion but maintaining the spherical shape of liposomes. Fluorescence polarization analysis revealed that the interaction of chitosan with liposomal membrane, such as electrostatic and hydrophobic interaction, restricted the motion freedom of lipid molecules and enhanced their ordering at the polar headgroup region and hydrophobic core of the membrane. These rigidifying effects were directly response for the stabilization of carotenoid-loaded liposomes against heating, gastrointestinal stress and centrifugal sedimentation. In addition, the encapsulating and retaining ability of chitosomes highly depended on the molecular structures of carotenoids. That is, the liposomal membrane was able to protect β-carotene and lutein to a higher level than lycopene and canthaxanthin. These results may contribute to the development of chitosomes as potential candidates for an efficient delivery of bioactive compounds in nutraceutical and functional foods.