The puckering transition of 4-substituted proline residues by electron-withdrawing groups, i.e., 4(R)-hydroxy-L-proline (Hyp) and 4(R)-fluoro-L-proline (Flp) residues, with trans and cis prolyl peptide bonds was studied by adiabatic optimizations along the torsion angle chi1 of the prolyl ring at the HF/6-31+G(d) level. By analyzing the potential energy surface and local minima, it is observed that the puckering transition of the prolyl ring for Hyp and Flp residues proceeds from a down-puckered conformation to an up-puckered one through the transition state with an envelope form having the N atom at the top of envelope and not a planar one for both trans and cis conformers, which is the same as found for the unsubstituted proline residue. At HF/6-31+G(d) and B3LYP/6-311++G(d,p) levels, the structures of the backbone and prolyl ring for local minima of Ac-Hyp-NHMe and Ac-Flp-NHMe are quite similar to those of Ac-Pro-NHMe. However, the relative stability of the up-puckered conformation to the down-puckered one is increased for Ac-Hyp-NHMe with the cis imide bond and for Ac-Flp-NHMe with the trans and cis imide bonds. In particular, the 4(R)-substitution by hydroxy and fluorine groups has brought some structural changes in the prolyl ring of the transition states and the changes in barriers for the puckering transition. The puckering transitions for Ac-Hyp-NHMe and Ac-Flp-NHMe are proven to be predominantly electronically driven by analyzing the electronic and enthalpic contributions to the barriers, as seen for Ac-Pro-NHMe.