Nicotine, the principal alkaloid in tobacco, exhibits significant central nervous system activity and induces a wide array of physiological effects. In addition to its well-documented role in tobacco dependence, previous studies have suggested that nicotine also has diverse pharmacological properties. These include alleviating symptoms associated with Parkinson's disease, potentially reducing the risk of Alzheimer's disease, mitigating oxidative stress, as well as anti-inflammatory and anxiolytic effects. Neuroscientists frequently use an array of molecular biology techniques to elucidate the mechanisms responsible for the effects of nicotine on the central nervous system. However, disease onset is invariably accompanied by metabolic dysfunction, and organisms often exhibit complex and unpredictable responses to pharmacological stimuli. As a bioactive alkaloid with potent pharmacological properties, nicotine is able to cross the blood-brain barrier and induce brain-compound changes, which serves as the basis for its effects on the central nervous system. Consequently, examining the extensive impact of nicotine exposure on endogenous metabolites and metabolic pathways in the brain is an indispensable step toward providing a more robust foundation for understanding the complex physiological effects of nicotine. In this study, an ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) metabolomic-analysis method was established to systematically examine the effects of repeated nicotine exposure on endogenous metabolites in mouse brains. Two chromatographic systems fitted with Acquity UPLC BEH HILIC (150 mm×2.1 mm, 1.7 μm) and BEH C18 (150 mm×2.1 mm, 1.7 μm) columns were used to determine the nicotine present in samples. As a result, the established UHPLC-MS/MS method identified a total of 759 endogenous metabolites. Compared with the saline group, nicotine exposure resulted in 575 significantly different metabolites, with 434 metabolites down-regulated and 141 up-regulated. Further pathway-enrichment analysis using the Kyoto Encyclopedia of Genes and Genomes (KEGG) revealed that nicotine exposure primarily affects essential-amino-acid, lipid, nucleotide, carbohydrate, cofactor, and vitamin metabolism, as well as other amino-acid metabolic pathways in the brain. Although non-targeted metabolomics can simultaneously detect and analyze all small-molecule metabolites in an unbiased manner, accurately capturing metabolite changes in specific brain regions is challenging when dealing with complex brain-tissue systems. Targeting the aggregation of material bases and the delivery of precision treatment to certain brain regions is expected to be significant for the targeted therapy of central nervous system diseases. Airflow-assisted desorption electrospray ionization mass spectrometry imaging (AFADESI-MSI) was further used to directly visualize the nicotine-induced distributions and variations of differentially expressed metabolites in various brain regions, which revealed that nicotine exposure leads to the significant downregulation of choline, serine, aspartate, and malate levels throughout the brain. Specifically, taurine, acetylcholine, and adenosine levels were notably affected in the cortical, hippocampal, and striatal regions, respectively. Essential-amino-acid metabolism was most affected by nicotine, with lipid metabolism found to be the next-most affected pathway. These metabolic pathways predominantly affected the cortical region, whereas the striatum, hippocampus, thalamus, and cerebellum were affected to varying degrees. These findings provide novel experimental evidence that enhances our understanding of metabolic biomarkers associated with nicotine exposure.