Abstract: Influences of cholesterol on N,N-dilauryl chitosan (DLCS) monolayer and corresponding vesicle property were studied. Experimental results indicated that the properties of N,N-dilauryl chitosan monolayer and corresponding vesicle were evidently changed by introducing cholesterol in matrix. Mixed monolayers with lower fraction of cholesterol were less rigid and compressible than pure DLCS monolayer. As the content of cholesterol increasing, however, the rigidity and compressibility of mixed monolayer were increased. In parallel, both the rate of drug-release and the equilibrium drug-release ratio of the vesicles were increased with lower cholesterol content, and they could be decreased with increasing the cholesterol content. The equilibrium drug-release ratio of the vesicle was decreased with increasing the compressibility of corresponding material monolayer, showing a linear relationship. Experimental results provide a possible way for preparing DLCS vesicle with controlled drug-release behavior by adjusting content of cholesterol in vesicle matrix.