Obesity and insulin resistance are characterised by altered metabolism and function in various tissues. In the liver, obesity contributes to liver-related metabolic diseases including insulin resistance and non-alcoholic fatty liver disease (Kitade et al., 2017). The ob/ob mouse, which produces a truncated inactive leptin protein, is widely used as a model of obesity-induced metabolic disease (Drel et al., 2006). Whilst the physiological phenotype of ob/ob mice has been well characterised, the underlying alterations in protein content and function remain relatively understudied. Using a deep-proteomic approach, we have identified divergent proteins and pathways that may underpin the mechanistic basis of obesity-induced liver dysfunction. Liver samples were harvested from four-month old ob/ob mice and lean littermates (n = 4 per group, C57BL/6J background) following a four-hour fast. Liver samples were lysed in a 4% SDS buffer and processed according to the MED-FASP protocol using Lys-C and trypsin. Peptides were separated on an Easy nano-flow HPLC system coupled to a LTQ Orbitrap mass spectrometer (HFX) via a nanoelectrospray source (Thermo Fisher Scientific). MS and MS/MS spectra were acquired in a data-dependent manner and analysed using MaxQuant software. Downstream data analysis was performed in Perseus software using label-free quantification (LFQ) intensities. Proteomics analysis of liver from lean and ob/ob mice led to the quantification of 5551 proteins. 330 proteins were differentially regulated with obesity, of which 172 and 158 proteins were up- or downregulated, respectively, in ob/ob mice. Gene Ontology annotations related to fatty acid metabolism were upregulated in the liver of ob/ob mice as well as the KEGG pathways PPAR signalling and peroxisome, indicating an adaptation to excess fatty acid availability. Furthermore, Gene Ontology cellular components related to the extracellular matrix were also upregulated in obese livers. Conversely, cellular component terms related to the endoplasmic reticulum were enriched in the downregulated proteins. The cytochrome p450 family, involved in the metabolism of arachidonic acid (a membrane phospholipid) and lipocalins, specifically the major urinary protein (MUP) isoforms, were also significantly downregulated in ob/ob liver. Overall, we provide a deep-proteomic analysis of liver from ob/ob and wild-type mice, identifying known and emerging regulators of insulin sensitivity within the liver. Further analyses will provide additional mechanistic insight into the development of liver dysfunction during obesity, in particular into the dysregulation of the extracellular matrix.