The mechanism of lipid flip-flop motion is important for maintaining the asymmetric distribution of lipids in a biological membrane. To explore the energetics and mechanism of passive cholesterol flip-flop and its dependence on chain saturation, we performed two-dimensional umbrella sampling simulations in DPPC, POPC, and DAPC (dipalmitoyl-, palmitoyloleoyl-, and diarachidonylphosphatidylcholine) and used the string method to identify the most probable flip-flop paths based on the two-dimensional free energy maps. The resulting paths indicate that cholesterol prefers to tilt first and then move to the bilayer center where the free energy barrier exists. The barrier is lower in DAPC than in DPPC or POPC, and the calculated flip-flop rates show that cholesterol flip-flop in a poly-unsaturated bilayer is faster than in more saturated bilayers. The free energy barrier results from the unfavorable enthalpic contribution arising from cholesterol-water/lipid interactions and the favorable entropic contribution due to increased lipid dynamics. While the cholesterol-water interaction has similar contributions to the barrier due to desolvation of the cholesterol hydroxyl group in all lipids, the cholesterol-lipid interaction has a much lower barrier in DAPC than in DPPC or POPC, resulting in the lower free energy barrier in DAPC.