The linear ubiquitin (Ub) chain assembly complex (LUBAC) is an E3 ligase that 
specifically assembles Met1-linked (also known as linear) Ub chains that 
regulate nuclear factor κB (NF-κB) signaling. Deubiquitinases (DUBs) are key 
regulators of Ub signaling, but a dedicated DUB for Met1 linkages has not been 
identified. Here, we reveal a previously unannotated human DUB, OTULIN (also 
known as FAM105B), which is exquisitely specific for Met1 linkages. Crystal 
structures of the OTULIN catalytic domain in complex with diubiquitin reveal 
Met1-specific Ub-binding sites and a mechanism of substrate-assisted catalysis 
in which the proximal Ub activates the catalytic triad of the protease. Mutation 
of Ub Glu16 inhibits OTULIN activity by reducing kcat 240-fold. OTULIN 
overexpression or knockdown affects NF-κB responses to LUBAC, TNFα, and 
poly(I:C) and sensitizes cells to TNFα-induced cell death. We show that OTULIN 
binds LUBAC and that overexpression of OTULIN prevents TNFα-induced NEMO 
association with ubiquitinated RIPK1. Our data suggest that OTULIN regulates 
Met1-polyUb signaling.