Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a major class of peptide natural products that often contain noncanonical amino acids and structural motifs with promising potential as drug leads. One unique structural unit found in RiPPs is the C-terminal S-[(Z)-2-aminoethenyl]-D-cysteine (AviCys) or (2S,3S)-S-[(Z)-2-aminoethenyl]-3-methyl-D-cysteine (AviMeCys). Avi(Me)Cys-containing RiPPs usually exhibit potent antimicrobial or anticancer activities, which strictly require the presence of the C-terminal AviCys motifs. Despite the potential of Avi(Me)Cys-containing RiPPs as drug leads, lack of synthetic methods and biosynthetic systems to access these type of cyclic peptides impede the application of Avi(Me)Cys-containing peptides in medicinal chemistry. In this review, we summarize the current understanding of the biosynthesis of Avi(Me)Cys-containing peptides and the progress made in the development of chemical methods to synthesize Avi(Me)Cys motifs and derivatives. This review contains two following major sections: ① The biosynthetic process of Avi(Me)Cys motifs in the different families of Avi(Me)Cys-containing RiPPs, including lanthipeptides, lipolanthines, linaridins and thioamitides, are introduced with three essential enzymatic steps: first, a cysteine decarboxylase oxidatively decarboxylated the C-terminal cysteine, generating a highly reactive enethiol; subsequently, distinct enzymes catalyze the dehydration of a serine/threonine (Ser/Thr) residue or the dethiolation of a Cys residue in the precursor peptide by incorporating a dehydroalanine (Dha) or dehydrobutyrine (Dhb) residue; finally, a putative cyclase catalyzes the Michael-type addition between the enethiol group and a Dha/Dhb residue to yield the Avi(Me)Cys motif. Detailed enzymatic investigation of these biosynthetic steps are introduced. ② The chemical synthesis of the Avi(Me)Cys building block and their analogues via diverse synthetic methodology, including the radical thiol-yne coupling, the oxidative decarboxylation/decarbonylation, and the condensation of amides with acetals. Overall, further elucidation of the complete biosynthetic pathway for Avi(Me)Cys motifs in related RiPPs, along with advancements in the chemical synthesis of Avi(Me)Cys-containing natural product peptides, will facilitate the effective utilization of these bioactive peptide natural products.