Recent experiments have shown that incorporating polyproline segments into arginine (R)–rich cell-penetrating peptides (CPPs) markedly enhances their membrane penetration. To elucidate the molecular basis of this effect, we designed a polyproline-containing R-rich peptide, P9R9, and investigated the role of proline residues in membrane translocation using molecular dynamics (MD) simulations. By employing the weighted ensemble approach, we reveal how the polyproline segment reshapes the spontaneous translocation mechanism of R-rich peptides. Notably, penetration efficiencies predicted by static in silico models differ substantially from those observed in MD simulations, highlighting mechanistic insights accessible only through dynamic simulations and beyond the reach of static modeling.