Thioamitides are apoptosis-inducing ribosomally synthesized and post-translationally modified peptides (RiPPs) with substantial post-translational modifications (PTMs), whose biosynthetic details remain elusive. We reconstituted their key PTMs through in vitro enzymatic reactions and gene coexpressions in E. coli and rigorously demonstrated the order of those modifications. Notably, thioamitide biosynthesis involves N- to C-terminal thioamidations and employs both leader-dependent and leader-independent reactions followed by leader removal by successive degradation. Our study provides a comprehensive overview of thioamitide biosynthesis and lays the foundation for thioamitide engineering in E. coli.