Wood-feeding arthropods incorporated specific microbial groups into their gut to expedite woody biomass decomposition and may contain the keys for the development of energy-efficient strategies for second-generation biofuels production. The wood-feeding beetle Odontotaenius disjunctus is known for its ability to ingest large amounts of woody biomass (1 kg/year/beetle), and a morphologically differentiated gut with steep radial oxygen gradients. Infrared analyses, ¹³C-thermochemolysis, and carbon and nitrogen bulk chemistry analyses demonstrated that cellulose and xylan depolymerization occurs in the gut together with lignin side chain oxidation and nitrogen accumulation. Our multi-omics approaches show that microbial communities are segregated within O. disjunctus’ gut regions, with the midgut (MG) and posterior hindgut (PHG) harboring similar microbiomes although separated by the anterior hindgut (AHG). We reconstructed 17 bacterial genomes from metagenomes and demonstrated that their localization within the gut corresponds with trait variation. Metagenomics and metaproteomics identified the MG as the key region for lignocellulose depolymerization and the AHG as the site for fermentation, methanogenesis, homoacetogenesis, and nitrogen fixation. Synchrotron X-Ray microCT imaging of intact beetles indicates that gut wall thickness likely plays an important role in driving the thermodynamics of these processes. Significant H₂ accumulation occurs in the AHG favoring acetogenesis over methanogenesis as a sink for hydrogen. Depolymerization continues in the PHG with a strong signal of further xylan depolymerization and xylose fermentation. Overall, these data illustrate how gut structural compartmentalization and assembly of microbial functional groups combine to enable lignocellulose deconstruction and the subsistence of this beetle on a low nutrient diet.