OTU deubiquitinases regulate diverse cellular processes by cleaving specific polyubiquitin chain linkages. While OTU deubiquitinases from bacteria, viruses, and animals are extensively characterized, OTU deubiquitinases from remain poorly studied. Arabidopsis thaliana, the model plant, have twelve OTU deubiquitinases, with no available structural information, defined polyubiquitin chain preferences, or identified physiological substrates. Here, we present a comprehensive molecular characterization of the complete Arabidopsis OTU deubiquitinase family through integrated structural, biochemical, and proteomic approaches. We determined crystal structures of apo atOTU1 and atOTU7, along with ubiquitin-bound complexes atOTU1:Ub2, atOTU2:Ub, and atOTU4:Ub, revealing the molecular basis for polyubiquitin recognition. Systematic biochemical analysis defined the polyubiquitin linkage specificity and kinetic parameters for all twelve atOTUs, while global interactome mapping identified the substrate networks for each family member. This comprehensive dataset reveals how atOTUs regulate distinct plant signaling pathways through their differential substrate recognition and provides a foundational resource for understanding ubiquitin-mediated regulation in plant biology. Our work establishes the molecular framework for plant OTU deubiquitinase function and enables targeted investigation of ubiquitin signaling in plant development and stress responses.