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Abstract: gastric cancer (GC) is one of the most common gastrointestinal tumors. As a newly discovered type of non-coding RNAs, transfer RNA (tRNA)-derived small RNAs (tsRNAs) play a dual biological role in cancer. Our previous studies have demonstrated the potential of tRF-23-Q99P9P9NDD as a diagnostic and prognostic biomarker for GC. In this work, we confirmed for the first time that tRF-23-Q99P9P9NDD can promote the proliferation, migration and invasion of GC cells in vitro. The dual luciferase reporter gene assay confirmed that tRF-23-Q99P9P9ND could bind to the 3'UTR site of acyl-CoA dehydrogenase short/branched chain (ACADSB). In addition, ACADSB could rescue the effect of tRF-23-Q99P9P9NDD on GC cells. Next, we used Gene Ontology, the Kyoto Encyclopedia of Genes and Genomes, and Gene Set Enrichment Analysis to find that downregulated ACADSB in GC may promote lipid accumulation by inhibiting fatty acid catabolism and ferroptosis. Finally, we verified the correlation between ACADSB and 12 ferroptosis genes at the transcriptional level, as well as the changes in reactive oxygen species levels by flow cytometry. In summary, this study proposes that tRF-23-Q99P9P9NDD may affect GC lipid metabolism and ferroptosis by targeting ACADSB, thereby promoting GC progression. It provides a theoretical basis for the diagnostic and prognostic monitoring value of GC and opens up new possibilities for treatment.