Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics. [Display omitted] • First structure of catalytically active, human Taspase1 has been resolved • The C-terminal region of Taspase1 α subunit adopts an "open-state" conformation • Deletion of the long C-terminal fragment abolishes Taspase1 catalytic activity Nagaratnam et al. solved the structure of the active Taspase1, a protease implicated in human cancers, and discovered a previously unresolved region is essential for the enzymatic activity and adopts a long helical conformation. Our study provides the basis for disruption of this fragment as potential strategy for anticancer drug design. [ABSTRACT FROM AUTHOR]