BACKGROUND AND STUDY AIMS: Nicotinamide N-methyltransferase (NNMT) is aberrantly expressed in tumors and is implicated in the progression and chemoresistance of cancers. This project attempts to explore the specific molecular mechanism by which NNMT enhances 5-fluorouracil (5-FU) resistance in gastric cancer (GC). MATERIALS AND METHODS: By bioinformatics analysis, the expression of NNMT in GC was analyzed and its relationship with patients' prognoses was examined. The signaling pathway enriched by NNMT was analyzed by the Kyoto Encyclopedia of Genes and Genomes (KEGG). Western blot (WB) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) were employed to measure the mRNA and protein expression of NNMT in normal gastric epithelial cells and GC cells. CCK8 was employed to measure cell viability and the IC RESULT: NNMT was greatly upregulated in GC tissues and cells, exhibiting a negative linkage with patients' prognoses. Knocking down NNMT remarkably repressed the vitality, proliferation, anti-apoptotic ability, migration, and invasion of GC cells but elevated the sensitivity of cancer cells to 5-FU. However, overexpression of NNMT inhibited H3K9 methylation by reducing the universal methyl donor SAM, activated the PI3K/AKT pathway, facilitated GC malignant progression, and triggered resistance to 5-FU. CONCLUSION: Upregulation of NNMT expression in GC cells can induce 5-FU resistance by repressing the activation of PI3K/AKT through the inhibition of histone methylation.