To investigate whether Dendrobium officinale polysaccharide (DOP) ameliorates bile acid-induced gastric intestinal metaplasia (GIM) via the histone deacetylase 3 (HDAC3)/hepatocyte nuclear factor 4α (HNF4α) pathway, a bile acidinduced GIM model was established using 100 μmol•L-1 chenodeoxycholic acid (CDCA) (cell viability 91.11%) and treated with DOP at concentrations of 400, 200, and 100 μg•mL-1. Morphological changes were observed, and cell proliferation was assessed via EdU staining. RT-PCR and Western blot were used to evaluate mRNA and protein expressions of Takeda G protein-coupled receptor 5 (TGR5), Farnesoid X receptor (FXR), HDAC3, caudal type homeobox gene 2 (CDX2), mucin 2 (MUC2), and HNF4α. An HDAC3-overexpressing GIM model was constructed to examine HDAC3 regulation of HNF4α using Western blot and co-immunoprecipitation (CO-IP). Results demonstrated that DOP exhibited no cytotoxicity toward GES-1 cells, whereas CDCA concentrations exceeding 100 μmol•L-1 significantly impaired cell viability. Although DOP (100~400 μg•mL-1) showed negligible effects on cell viability or proliferation in the CDCA-induced GIM model, it effectively maintained the normal phenotype of GES-1 cells. Furthermore, DOP significantly downregulated gene and protein expressions of CDX2, MUC2, HNF4α, TGR5, FXR, and HDAC3. In the HDAC3-overexpressing GIM model, HDAC3 and HNF4α protein levels increased by 52.52% and 62.94%, respectively, which were subsequently reduced by DOP intervention. CO-IP confirmed a direct interaction between HDAC3 and HNF4α. Collectively, these findings indicate that DOP alleviates bile acid-induced GIM by suppressing the expressions of histone deacetylase, bile acid receptors, and intestinal metaplasia markers via the HDAC3/HNF4α pathway, providing a theoretical foundation for treating gastric precancerous lesions.