Peptidoglycan (PG) is a fundamental component of bacterial envelopes, and it is processed by a wide range of hydrolases during cell growth and division1–3. In contrast, in Archaea only one clade -regrouping the Methanobacteriales and the Methanopyrales- possess cell walls made of PG (arcPG)4–6. The structure of arcPG (also known as pseudomurein) was resolved in the 80s by purely chemical methods7–11. Despite its overall similarity to bacterial PG, arcPG has striking structural differences, notably it lacks N-Acetylmuramic acid (MurNAc) but instead has N-Acetyltalosaminuronic acid (TalNAc), and GlcNAc and TalNAc are connected via unique (1,3) glycosidic bonds6. These peculiarities make arcPG insensitive to muramidases active on bacterial PG such as lysozyme or mutanolysin. To date, no glycosyl hydrolase cleaving arcPG has been described. Here we report the discovery and characterization of TalA, the first enzyme with Talosaminidase activity from Methanobrevibacter smithii, the most abundant species of walled methanogenic archaea from the human gut. It is a dual function enzyme, cleaving both the peptide stem and glycan strand. The identification of TalA allowed us to revise the chemical structure of arcPG, revealing the presence of a previously undescribed modification on the Talosaminuronic acid moiety which appears to be a conserved feature in all walled archaea. Phylogenetic analysis and taxonomic distribution show that TalA homologues are specific to walled archaea, and we experimentally demonstrate that M. smithii TalA is active on arcPG from diverse archaea. Finally, quantitative and high-resolution imaging of TalA subcellular localization indicates it is the main hydrolase involved in septum cleavage in walled archaea, also confirmed by the phenotype of a TalA mutant. Our results identify a novel enzyme involved in the archaeal cell cycle and establish an essential tool to further study the biology of walled archaea both in vitro and in vivo. These findings also open important perspectives toward the development of mitigation strategies for methanogens in man-made and natural environments, including the gastrointestinal tract.