<p>Esophageal squamous cell carcinoma (ESCC) is a highly prevalent malignancy in China. Understanding its metabolic heterogeneity is of great significance for improving the precision of diagnosis and treatment. This study innovatively employed an adult zebrafish xenograft model, combining the spatial metabolomics and in situ derivatization method for fatty acid peroxidation products (FAPPs) based on air flow-assisted desorption electrospray ionization–mass spectrometry imaging (AFADESI-MSI) technology, to elucidate the metabolic patterns associated with ESCC and its differentiation grade. Spatial metabolomic data demonstrated that abundance alterations of glycerophospholipids and acetylated amino acids were statistically correlated with ESCC tumor formation. Levels of lysophospholipids and arachidonic acid-associated metabolites tended to increase with the progressive malignant differentiation of tumor lesions. Profiling of FAPPs showed elevated short- and medium-chain species accompanied by decreased long-chain counterparts in tumor-bearing zebrafish, which may be linked to tumor-initiated alterations in oxidative stress. Specifically, short- and medium-chain FAPPs were enriched in well-differentiated ESCC tissues, while long-chain FAPPs predominated in poorly differentiated tumors, suggestive of disparate lipid peroxidation patterns in ESCC cells at different differentiation stages. Furthermore, distinct signature metabolites were found to be specifically accumulated in the brain providing experimental clues that ESCC may remotely disturb the central nervous system metabolism of host animals. This research uncovers both tumor-induced and differentiation-dependent metabolic heterogeneity of ESCC, and validates whole-body MSI as a transformative approach to unravel tumor-host metabolic interactions.</p> Graphical abstract <p></p>