The NF-κB regulator A20 antagonises IKK activation by modulating Lys63-linked polyubiquitination of cytokine receptor associated factors including TRAF2/6 and RIP1. Here we describe the crystal structure of the N-terminal Ovarian Tumour (OTU) deubiquitinase domain of A20, which differs from other deubiquitinases but shares the minimal catalytic core with Otubain-2. Analysis of conserved surface regions allows prediction of ubiquitin binding sites for the proximal and distal ubiquitin molecules. Structural and biochemical analysis suggests a novel architecture of the catalytic triad, which might be present in a subset of OTU domains including Cezanne and TRABID. Biochemical analysis shows a preference of the isolated A20 OTU domain for Lys48-linked tetraubiquitin in vitro suggesting that additional specificity factors might be required for the physiological function of A20 in cells.