Cyclic disulfide-rich peptides have attracted interest in drug design and development. The most common approach for cyclic peptide production is solid-phase peptide synthesis, which relies on expensive amino acid building blocks, hazardous reagents and solvents, thus making large-scale production costly and environmentally damaging. Here, we present a yeast-based production system utilizing asparaginyl endopeptidase-mediated head-to-tail ligation to produce cyclic disulfide-rich peptides. Using this platform, an engineered functional α- conotoxin has been successfully produced in its native “globular” conformation. Scale-up production in 5-liter bioreactors, allowed high levels of production with yields of cyclic peptide reaching 85-97 mg L−1 of culture, surpassing the highest yield obtained by any other recombinant expression system. The production system established in this study provides a new approach for the manufacture of cyclic disulfide-rich peptides, reducing the significant environmental burden caused by commonly used chemical synthesis.