Among various peptide modification strategies, thioamide substitution by replacing the carbonyl oxygen atom of an amide bond with a sulfur atom constitutes an invaluable tool for chemical biology, for use in peptide drug discovery and protein structure-function studies. However, the thioamide substitution effect has not been well studied because of the lack of synthetic methods for site-specifically incorporating a thioamide bond into a peptide backbone, particularly introducing multiple thioamide substitutions into peptide on a solid support. Herein, we report a highly efficient method for incorporating a thioamide bond into the peptide backbone in a site-specific manner by employing α-thioacyloxyenamides, which are formed from the addition of N-protected monothioamino acids and ynamides, as novel thioacylating reagents in solid phase peptide synthesis. This method is amenable for 19 of 20 proteinogenic amino acids, His being the exception. One to multiple thioamide substitutions could be incorporated into a growing peptide with no epimerization or a low level of epimerization. By using this method, a fully thioamide-substituted hexapeptide containing up to five continuous thioamide bonds could be synthesized smoothly. This synthetic methodology will spur the application of the thioamide substitution tool for protein engineering and peptide drug discovery.