For the development of orally available liposomes, understanding the interaction of liposomes with the intestinal mucosa is important. An unstirred water layer (UWL) on the intestinal epithelium surface is a considerable permeability barrier for lipophilic drugs. Therefore, the effects of an UWL on liposome transport across intestinal epithelial cells must be elucidated. We evaluated the effects of the surface charge, particle size, and polyethylene glycol (PEG) modification of liposomes on their association with Caco-2 cells across an UWL. When the association of cationic liposomes with Caco-2 cells was evaluated under a reduction in UWL thickness by shaking, the uptake and/or amount of surface-bound cationic liposomes in cells was increased significantly in a shaking rate-dependent manner. The uptake and/or amount of surface-bound neutral liposomes were increased only at the highest shaking rate. No significant differences in the cellular association of anionic liposomes and PEG-modified liposomes were observed with or without shaking. The association of large liposomes with Caco-2 cells was affected considerably by an UWL compared with that of small liposomes. These results suggest that an UWL affects the surface binding and subsequent uptake of liposomes in Caco-2 cells according to their particle size, surface charge, and PEG modification.