Proteases are ubiquitous and >400,000 putative sequences are found in databases. In contrast, ligases which are peptide-forming enzymes catalyzing the reverse reaction are rare. Thus far only four are identified. Recently, we reported the discovery of butelase 1, a versatile and multi-purpose ligase which is specific for the C-terminal Asn/Asp (Asx) ligation. Butelase 1, isolated from Clitoria Ternatea of the legume family, requires an Asn/Asp (Asx) residue as the recognition site and a sorting signal such as a tripeptide motif Asx-HisVal at the C-terminus of a peptide or protein substrate with HisVal dipeptide as a leaving group (Figure 1) [1]. Butelase 1 accepts most amino acids as a nucleophile to form an Asx-Xaa bond without a trace of the sorting signal. Among the known ligases including sortase A, PATG and PCYC1, butelase 1 is the fastest ligase with catalytic efficiencies as high as 1,300,000 M s. These favorable properties bode well for applications of butelase 1 for ligation, macrocyclization and labeling of peptides, proteins and live cells. Here, we present our recent work of butelase 1 in peptide macrocyclization using sunflower trypsin inhibitor and histatin as examples.