Molecular dynamics simulation is applied to investigate the bilayer properties of a novel catanionic vesicle composed of an ion pair amphiphile, hexadecyltrimethylammonium-dodecylsulfate (HTMA-DS), with cholesterol. Structural properties, such as molecular organization, orientation, and conformation, were analyzed from the resulting trajectory. Simulation results showed that cholesterol could induce both condensing and disordering effects on the rigid HTMA-DS bilayer. The condensing effect of cholesterol was ascribed to the maximizing contact between cholesterol ring and the neighboring hydrocarbon chains. Thus, the inserted cholesterol ring restrained the neighboring hydrocarbon chain segments from motion and increased the order of the neighboring hydrocarbon chains. However, the presence of cholesterol would increase the distance between head groups of HTMA-DS and induce a shift of DS(-) head groups toward the inside of the bilayer. This led to the protrusion of the HTMA(+) head groups and conformational disorder in the front segments of HTMA(+) hydrocarbon chains. In addition, the cholesterol-induced void in the hydrophobic core of the HTMA-DS bilayer increased the motion freedom of the terminal segments of the hydrocarbon chains. The cholesterol-induced space in the polar region and void in the nonpolar region of the bilayer led to a conformational disorder. With high cholesterol contents, the conformational disorder effect would overwhelm the condensing effect, resulting in the apparent disordering effect on the rigid HTMA-DS bilayer.