AIM: To explore the effect of lysine acetylation in relat- ed proteins on regulation of the proliferation of gastric cancer cells, and determine the lysine-acetylated pro- teins and the acetylated modified sites in AGS gastric cancer cells. METHODS: The CCK-8 experiment and flow cytometry were used to observe the changes in proliferation and cycle of AGS cells treated with trichostatin A (TSA). Real time polymerase chain reaction and Western blot- ting were used to observe expression changes in p21, p53, Bax, Bcl-2, CDK2, and CyclinD1 in gastric cancer cells exposed to TSA. Cytoplasmic proteins in gastric cancer cells before and after TSA treatment were im- munoprecipitated with anti-acetylated lysine antibodies, separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis gel and silver-stained to detect the proteins by mass spectrometry after removal of the gel. The acetylated proteins in AGS cells were enriched with lysine-acetylated antibodies, and a high-resolution mass spectrometer was used to detect the acetylated proteins and modified sites.RESULTS: TSA significantly inhibited AGS cell prolifera- tion, and promoted cell apoptosis, leading to AGS cell cycle arrest in G0/G1 and G2/M phases, especially G0/ G1 phase. p21, p53 and Bax gene expression levels in AGS cells were increased with TSA treatment duration; Bcl-2, CDK2, and CyclinD1 gene expression levels were decreased with TSA treatment duration. Two unknown protein bands, 72 kDa (before exposure to TSA) and 28 kDa (after exposure to TSA), were identified by silver- staining after immunoprecipitation of AGS cells with the lysine-acetylated monoclonal antibodies. Mass spec- trometry showed that the 72 kDa protein band may be PKM2 and the 28 kDa protein band may be ATP5O. The acetylated proteins and modified sites in AGS cells were determined.CONCLUSION: TSA can inhibit gastric cancer cell pro- liferation, which possibly activated signaling pathways in a variety of tumor-associated factors. ATP5O was ob- viously acetylated in AGS cells following TSA treatment.