Dendrobium huoshanense C. Z. Tang et S. J. Cheng is an important edible medicinal plant that thickens the stomach and intestines, and its active ingredient, polysaccharide, can have anti-inflammatory, immunoregulatory, and antitumor effects. However, the gastroprotective effects and potential mechanisms of Dendrobium huoshanense polysaccharides (DHP) remain unclear. An N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) induced human gastric mucosal epithelial cells (GES-1) damage model was used in this research, aiming to investigate whether DHP has a protective effect on MNNG-induced GES-1cell injury and its underlying mechanism based on the combination of multiple methods. DHP was extracted using water extraction and alcohol precipitation methods, and the proteins were removed using the Sevag method. The morphology was observed using scanning electron microscopy. A MNNG-induced GES-1cell damage model was developed. Cell viability and proliferation of the experimental cells were investigated using a cell counting kit-8 (CCK-8). Cell nuclear morphology was detected using the fluorescent dye Hoechst 33342. Cell scratch wounds and migration were detected using a Transwell chamber. The expression levels of apoptosis proteins (Bcl-2, Bax, Caspase-3) in the experimental cells were detected by Western blotting. Ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) was performed to investigate the potential mechanism of action of DHP. The CCK-8 kit analysis showed that DHP increased GES-1cell viability and ameliorated GES-1cell injury by MNNG. In addition, scratch assay and Transwell chambers results suggested that DHP improved the MNNG-induced motility and migration ability of GES-1cells. Likewise, the results of the apoptotic protein assay indicated that DHP had a protective effect against gastric mucosal epithelial cell injury. To further investigate the potential mechanism of action of DHP, we analyzed the metabolite differences between GES-1cells, GES-1cells with MNNG-induced injury, and DHP+MMNG-treated cells using UHPLC-HRMS. The results indicated that DHP upregulated 1-methylnicotinamide, famotidine, N4-acetylsulfamethoxazole, acetyl-L-carnitine, choline and cer (d18:1/19:0) metabolites and significantly down-regulated 6-O-desmethyldonepezil, valet hamate, L-cystine, propoxur, and oleic acid. DHP may protect against gastric mucosal cell injury through nicotinamide and energy metabolism-related pathways. This research may provide a useful reference for further in-depth studies on the treatment of gastric cancer, precancerous lesions, and other gastric diseases.