Peptides offer great promise as therapeutics, but their application is often limited by poor stability and oral availability. Cyclisation of the peptide backbone is one way of endowing peptides with favourable properties that overcome these limitations, but there are few molecular tools that are suitable for large scale and affordable production. Here, we report the recombinant expression of plant-derived asparaginyl endopeptidases (AEPs), a new class of peptide ligases. The activity of these enzymes was characterised using mass spectrometry and high performance liquid chromatography to track the enzymatic processing products of a variety of substrates. These enzymes functioned as efficient peptide ligases by coupling release of a C-terminal propeptide to the formation of a new amide bond. Furthermore, they can be used to create either a backbone cyclised peptide (intra-molecular ligation) or new linear peptides (inter-molecular ligation). Importantly, these enzymes can efficiently cyclise peptides that naturally occur in a cyclic form, as well as peptides that are naturally linear following the strategic addition of as few as three non-native residues. In addition, by subtle alteration of the reaction conditions, these enzymes can also function as inter-molecular ligases to introduce site-specific labels at the N- or C-termini. These enzymes apply limited structural and sequence requirements to their targets, making it likely they will find broad application as tools in protein engineering.