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The carnitine palmitoyltransferase (CPT) family is essential for fatty acid oxidation. Recently, we found that CPT1C, one of the CPT1 isoforms, plays a vital role in cancer cellular senescence. However, it is unclear whether other isoforms (CPT1A, CPT1B, and CPT2) have the same effect on cellular senescence. This study illustrates the different effects of CPT knockdown on PANC‐1 cell proliferation and senescence and MDA‐MB‐231 cell proliferation and senescence, as demonstrated by cell cycle kinetics, Bromodeoxyuridine incorporation, senescence‐associated β‐galactosidase activity, colony formation, and messenger RNA (mRNA) expression of key senescence‐associated secretory phenotype factors. CPT1C exhibits the most substantial effect on cell senescence. Lipidomics analysis was performed to further reveal that the knockdown of CPTs changed the contents of lipids involved in mitochondrial function …