The Tribbles family of pseudokinases recruit substrates—including metabolic proteins and transcription factors—to the COP1 ubiquitin ligase for ubiquitin-mediated protein degradation. CCAAT-enhancer binding protein (C/EBP) family transcription factors are a particularly relevant class of Tribbles-COP1 substrate in adipocyte and myeloid development, and acute myeloid leukaemia. We have solved several crystal structures of TRIB1, including an autoinhibited state and the TRIB1 pseudokinase in complex with the Tribbles recognition degron from C/EBPα. The structures show that TRIB1 undergoes a significant conformational change relative to its substrate-free structure to bind C/EBPα in a pseudo-substrate-like manner. Importantly, substrate-induced rearrangement of TRIB1 engages an allosteric network that links substrate recruitment, release of the COP1 binding-motif of TRIB1 and formation of the cognate COP1-TRIB1 ubiquitin E3 ligase. Such a cooperative model is consistent with biochemical experiments and molecular dynamics simulations. These findings offer a view of pseudokinase regulation with striking parallels to bona fide kinase regulation—via the activation loop and αC-helix—and raise the possibility of small molecules or binding proteins that target either the activation loop-in or loop-out conformations of Tribbles pseudokinases.