We conducted a time-dependent study of cholesterol (Ch) nucleation to investigate the effect of calcium ions in the moderate supersaturated bile. In taurocholate/lecithin (TC/L) bile at a TC to L ratio of 5.1, the presence of calcium ions enhanced the nucleation rate of Ch. Contrarily, we observed the delayed nucleation of Ch after~30 days in TC/L bile at a ratio of 2.0, regardless of the calcium ions. From the physical chemistry standpoint, the cholesterol activity (ChA T) is believed to be the driving force for Ch nucleation together with the sufficient nucleation sites. Hence, the micellar formation models along with the binding of TC monomers to calcium ions interpreted the timedependent results. Furthermore, a quasielastic light-scattering technique detected the formation of micelles and vesicles. In conclusion, the presence of calcium ions in TC/L bile at a high ratio enhances the vesicle appearance for nucleation sites and the high ChA T values for fast nucleation rate of Ch. However, an increase in the L concentration (i.e., low ratio bile) plays a significant role in the prevention of Ch gallstone formation, compared to the decrease in calcium ion concentration.