<p>Colorectal cancer (CRC) exhibits substantial metabolic heterogeneity, which plays a critical role in tumor progression and treatment response. However, systematic comparisons of metabolic profiles among widely used CRC cell lines are still lacking. Understanding these metabolic differences is essential for improving the translational relevance of preclinical studies. The study aims to conduct a comprehensive metabolomic analysis on three widely used CRC cell lines (HT-29, Caco-2, SW480) to identify their metabolic differences and explore their biological significance and origins. We conducted a metabolomic analysis using ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to measure intracellular and extracellular metabolites. Multivariate statistical approaches, including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA), were applied to identify metabolic variations. Pathway enrichment analysis was then performed to uncover dysregulated metabolic pathways. Our analysis revealed distinct metabolic profiles among the three CRC cell lines, with 25 differential metabolites identified. Pathway enrichment analysis highlighted three significantly dysregulated pathways: glycerophospholipid metabolism, ether lipid metabolism, and pantothenate and CoA biosynthesis. These findings demonstrate intrinsic metabolic heterogeneity at both intracellular and extracellular levels. These findings confirm intrinsic metabolic heterogeneity of the three CRC cell lines by intracellular and extracellular profiling, highlighting the importance of validating results across multiple models to address the limitations intrinsic to single cell lines in mechanistic and translational studies.</p>