Cholesterol is a key regulator of membrane biophysical properties, yet its role in modulating cell-penetrating peptide (CPP) translocation remains incompletely understood. Here, weighted ensemble molecular dynamics simulations were employed to characterize the translocation of the arginine-rich CPP R9 (RRRRRRRRR) across a model membrane composed of DOPC, DOPG, and cholesterol at a 4:1:1 molar ratio, with a cholesterol-free DOPC/DOPG bilayer used for comparison. As expected, cholesterol incorporation increased lipid tail ordering and membrane bending rigidity while reducing the area per lipid. These changes indicate a mechanically stiffer and more ordered membrane environment, which would generally be expected to impede peptide translocation. Nevertheless, potential of mean force calculations revealed a lower free-energy barrier for R9 translocation in the cholesterol-containing membrane than in the cholesterol-free bilayer. Analysis of peptide–water interactions along the translocation pathway further showed that R9 remained more highly hydrated within the cholesterol-containing membrane, reducing the energetic penalty of transferring its positively charged arginine residues into the hydrophobic bilayer interior. These results suggest that CPP translocation is governed by an interplay between membrane mechanics and peptide hydration: cholesterol-induced membrane stiffening tends to oppose peptide translocation, whereas enhanced local hydration of R9 favors its passage through the membrane. The balance between these competing effects reduces the free-energy barrier in the cholesterol-containing membrane. Our findings therefore highlight the coupled roles of membrane mechanics and peptide hydration in determining CPP translocation energetics and provide molecular-level insight into how cholesterol modulates the membrane permeation of arginine-rich peptides.