Nisin is a pentacyclic peptide antibiotic active against Gram-positive bacteria. Its thioether rings are formed by two enzymatic steps: nisin dehydratase (NisB)-mediated dehydration of serines and threonines followed by nisin cyclase (NisC)-catalyzed enantioselective coupling of cysteines to the formed dehydroresidues. Here, we report the in vivo activity of NisC to cyclize a wide array of unrelated and designed peptides that were fused to the nisin leader peptide. To assess the role of NisC, leader peptide fusions, secreted by Lactococcus lactis cells containing NisBT with or without NisC were compared. In hexapeptides, a dehydroalanine could spontaneously react with a more C-terminally located cysteine. In contrast, peptides containing dehydrobutyrines require NisC for cyclization. In agreement with in silico predictions NisC could efficiently cyclize the hexapeptides ADhbVECK and IDhbPGCK, but ADhbVWCE was not cyclized. Interestingly, NisC could efficiently catalyze the synthesis of peptides with intertwined rings and of a designed polyhexapeptide containing four thioether rings. Taken together the data demonstrate that NisC can be widely applied for the cyclization and stabilization of nonlantibiotic peptides.