Background and Aims The oncogene PIM1, encoding a constitutively active serine/threonine kinase, is upregulated in various tumors and closely associated with tumorigenesis and progression. Our previous studies have confirmed the oncogenic role of PIM1 in thyroid cancer, demonstrating its impact on tumor development, progression, and prognosis. This study was conducted to investigate the metabolic characteristics of papillary thyroid carcinoma (PTC) cells overexpressing PIM1 using metabolomics analysis, providing a basis for further exploring how PIM1 regulates metabolic processes in PTC.Methods A PIM1 overexpression plasmid was constructed, and a stably transfected PTC cell line (BCPAP) was established. The overexpression of PIM1 was verified by qRT-PCR and Western blot. Metabolomics analysis of PIM1-overexpressing BCPAP cells (PIM1-OE) and control cells transfected with an empty vector (NC) was performed using liquid chromatography-mass spectrometry (LC-MS). Differential metabolites and metabolic pathways were identified and screened through multivariate statistical analysis and the KEGG database. Bioinformatics analysis was conducted to explore the relationship between PIM1, associated metabolic pathways, and the prognosis of thyroid cancer patients.Results The mRNA and protein levels of PIM1 were significantly upregulated in the PIM1-OE BCPAP cells (both P<0.05). Metabolomics analysis identified changes in 41 intracellular metabolites in PIM1-OE cells, with 15 metabolites showing significant differences, including L-aspartic acid, succinic acid, L-threonine, L-tryptophan, betaine, 2-dehydropantothenate, 3-indoleacetonitrile, D-octopine, indole, N-acetylglutamic acid, creatine, pantothenic acid, uremic acid, N-acetyl-L-aspartic acid, and hydroxyphosphono-pyruvic acid (all P<0.05). KEGG database analysis revealed significant alterations in 31 metabolic pathways, with 4 pathways—alanine, aspartate and glutamate metabolism; tryptophan metabolism; glycine, serine, and threonine metabolism; and arginine and proline metabolism—being notably affected, showing decreased activity (all P<0.05). Bioinformatics analysis indicated that high PIM1 expression and reduced activity in the glycine, serine, and threonine metabolic pathways were associated with significantly decreased overall survival in thyroid cancer patients (both P<0.05).Conclusion PIM1 may influence multiple metabolic pathways by regulating the levels of various amino acids, including aspartic acid, succinic acid, and tryptophan, thereby altering the metabolic state of PTC and promoting its development and progression. Among these, changes in the glycine, serine, and threonine metabolic pathways may be closely associated with patient prognosis.