Non-alcoholic fatty liver disease (NAFLD) can progress from 'simple steatosis' (NAFL) to non-alcoholic steatohepatitis (NASH), cirrhosis and cancer. Currently the driver for this progression is not fully understood; in particular it is not known how NAFLD and its early progression affects the distribution of lipids in the liver, producing lipotoxicity and inflammation. Here we use dietary and genetic mouse models of NAFL and NASH, and translate results to humans, by correlating the spatial distribution of lipids in liver tissue with disease progression, using advanced mass spectrometry imaging technology. We identified several lipids with distinct zonal distributions in control and NAFL samples, whilst partial to complete loss of lipid zonation was found in NASH. In addition, we found increased hepatic expression of genes associated with remodelling the phospholipid membrane, release of arachidonic acid (AA) from the membrane, and production of eicosanoid species which promote inflammation and cell injury. Using immunohistochemistry, we further suggest that the zonal location of remodelling enzyme lysophosphatidylcholine acyl transferase 2 (LPCAT2) plays a role in the change in spatial distribution for arachidonic acid (AA)-containing lipids. This results in a cycle of AA-enrichment in pericentral hepatocytes, membrane release of AA and generation of pro-inflammatory eicosanoids, and may account for increased oxidative damage in pericentral regions in NASH. CONCLUSION: We have demonstrated that NAFLD is associated not only with lipid enrichment, but also to zonal changes of specific lipids and their associated metabolic pathways. This may play a role in the heterogeneous development of NAFLD. ; Other ; Medical Research Council (MRC). Horizon 2020 Framework Program of the European Union.