•Chronic alcohol exposure profoundly modifies lipidome profiling in PFC and striatum.•Three lipid classes, GP, GL and FA, take the most proportion of those modified lipidome.•The majority of altered lipids are synthesized in ER.•ER stress may account for the neurotoxicity of chronic alcohol caused disorders. Much efforts have been tried to clarify the molecular mechanism of alcohol-induced brain damage from the perspective of genome and protein; however, the effect of chronic alcohol exposure on global lipid profiling of brain is unclear. In the present study, by using Q-TOF/MS-based lipidomics approach, we investigated the comprehensive lipidome profiling of brain from the rats orally administrated with alcohol daily, continuously for one year. Through systematically analysis of all lipids in prefrontal cortex (PFC) and striatum region, we found that long-term alcohol exposure profoundly modified brain lipidome profiling. Notably, three kinds of lipid classes, glycerophospholipid (GP), glycerolipid (GL) and fatty acyls (FA), were significantly increased in these two brain regions. Interestingly, most of the modified lipids were involved in synthetic pathways of endoplasmic reticulum (ER), which may result in ER stress-related metabolic disruption. Moreover, alcohol-modified lipid species displayed long length of carbon chain with high degree of unsaturation. Taken together, our results firstly present that chronic alcohol exposure markedly modifies brain lipidomic profiling, which may activate ER stress and eventually result in neurotoxicity. These findings provide a new insight into the mechanism of alcohol-related brain damage.