Thioamide substitution has evolved into an important tool for the chemical biology of peptides and proteins.1 However, exploration of its full potential has been hampered by insufficient synthetic strategies for site-specific introduction of a thioamide bond to a peptide backbone. We recently discolsed that ynamides could be used as racemization-free coupling reagents for peptide synthesis.2 Herein, we reported a novel ynamide-mediated two-step thioamide bond formation strategy which could be used for site-selectively incorporating of a thioamide substitution into a peptide backbone by employing monothioamino acids as the sulfur source.3 The α-thioacyloxyenamide intermediates formed as the major product of the addition reactions of ynamides and monothiocarboxylic acids are stable and can be purified, characterized and stored. The appropriate balance between their activity and stability enables them to be effective thioacylating reagents to afford thiopeptide bonds under mild reaction conditions. Notorious issues such as racemization/epimerization and the use of toxic and smelly thionating reagents, which persecute thiopeptide synthesis for a long time, have been addressed. Some side chain functional groups of amino acids such as –OH, –CONH2, and the NH of indole are tolerated, rendering their protection during thiopeptide synthesis unnecessary. Importantly, the modular nature of the ynamide-mediated thioamid bond formation makes this strategy amenable to site-specific incorporating of a thioamide bond to peptide backbone not only in solution-phase peptide synthesis but also in solid-phase peptide synthesis.