Aristolochic　acid　I　(AAI)　as　one　of　the　major　aristolochic　acids　(AAs)　can　cause　progressive　aristolochic　acid　nephropathy　(AAN),　which　has　been　widely　investigated　since　the　early　1990s.　Besides　renal　diseases,　it　has　been　recently　revealed　that　AAI　can　induce　liver　damage.　In　this　study,　we　report　the　molecular　mapping　of　liver　tissue　sections　from　AAI-exposed　mice　using　atmospheric　pressure　matrix-assisted　laser　desorption/ionization　mass　spectrometry　(AP-MALDI　MS)　and　show　the　distinct　metabolic　alterations　when　compared　to　the　control　group.　We　first　used　renal　tissue　sections　to　evaluate　the　performance　of　AP-MALDI　MSI　in　spatial　discrimination　of　different　morphological　regions.　Then,　the　hepatic　tissues　from　the　AAI-induced　and　the　control　mice　were　analyzed,　displaying　rich　metabolic　profiles　from　both　groups.　Orthogonal　partial　least　squares-discriminant　analysis　(OPLS-DA)　is　used　to　show　complete　separation　of　the　two　groups.　A　machine　learning　algorithm–least　absolute　shrinkage　and　selection　operator　(lasso)　is　used　for　statistical　analysis　of　a　total　of　11,726　pixels　of　imaging　data　extracted　from　3　normal　liver　and　3　AAI-exposed　liver　tissue　sections,　generating　a　classifier　with　high　accuracy　(99.81%).　In　total,　16　m/z　values,　including　small　metabolites　and　lipid　species,　are　selected　to　discriminate　AAI-exposed　liver　tissues.　Finally,　we　explore　the　potentially　impacted　pathways　using　metabolomics　pathway　analysis　(MetPA),　indicating　multiple　metabolic　pathway　alterations　including　taurine　and　hypotaurine　metabolism,　glycerophospholipid　metabolism,　D-Glutamine　and　D-glutamate　metabolism,　and　arachidonic　acid　metabolism,　which　provides　new　insights　in　AAI-induced　hepatotoxicity.　©　2022