Polyproline exists predominately as the all-cis polyproline I (PPI) helix in aliphatic alcohols, whereas the all-trans polyproline II (PPII) helix is favored in aqueous solutions. Previous ion mobility spectrometry-mass spectrometry (IMS-MS) work demonstrates that the gas-phase conformations of polyproline ions can be related to the corresponding PPI and PPII helices in solution [J. Phys. Chem. B 2004, 108, 4885]. Here, we use IMS-MS to examine the detailed intermediate steps associated with the process of Polyproline-13 (Pro13) conversion from the PPI helix to the PPII helix upon solvent exchange. Collision cross section distributions of Pro13 [M + 2H](2+) ions obtained at different transition times indicate the presence of two major conformers, identified as the PPI and PPII helices, and six conformers that appear as subpopulations of polyproline. Further analysis shows a transition mechanism with sequential cistrans isomerizations followed by a parallel process to establish PPII and two smaller subpopulations at equilibrium. Temperature-dependent studies are used to obtain Arrhenius activation parameters for each step of the mechanism, and molecular dynamics simulations provide insight about the structures of the intermediates. It appears that prolines sequentially flip from cis to trans starting from the N-terminus. However, after the first few transitions, possible steps take place at the center of the peptide chain; subsequently, several pathways appear to be accessible at the same time. Our results reflect the existence of stable subpopulations in polyprolines and provide new insight into the structural changes during the transition process of polyproline peptides converting from PPI to PPII in aqueous solution