The saturated ring and secondary amine of proline exists equilibria between ring puckers and peptide bond isomers. These conformational equilibria can be modulated by substitution on the proline ring. Implanting an electron-withdrawing group on the 4S position of proline prefers a Cγ-endo pucker and a cis peptide bond due to stereoelectronic effects, and favors polyproline I (PPI) helices rather than polyproline II (PPII) helices by preorganization. 4-Thiaproline (Thp) favors an endo ring pucker and a cis peptide bond, and here we incorporated Thp into polyproline peptides to explore its effects on the peptide conformation. We synthesized a series of peptides, including P5ThpP5, P11, Thp7, and P7, and characterized their structures by CD spectroscopy. The results show that Thp substitutions not only destabilize PPII helices but also decrease the tendency to form PPI helices. The density functional theory (DFT) analysis on single residues (Ac-Pro-OMe and Ac-Thp-OMe) and short polyproline structures (Ac-P2-Thp-P2-OMe and Ac-P5-OMe) reveals that the energy difference between two ring puckers is small for Thp and Thp only slightly favors the endo pucker. Such a small energy difference could lead to the coexistence of PPI and PPII in solution for Thp-containing peptides. Natural bond orbital (NBO) analysis on Ac-Pro-OMe and Ac-Thp-OMe also shows that the backbone n→π* interaction in Ac-Thp-OMe is smaller than that in Ac-Pro-OMe, consistent with our experimental data. Our data demonstrate that although Thp possesses a structure similar to proline, it has a significant impact on polyproline conformation.