The chemical characterization and protective role against ethanol-induced gastric ulcerated rats of a polysaccharide fraction from Bletilla striata (BSP) collected by ultrafiltration membrane approach were evaluated. This BSP faction was consisted of mannose and glucose at a molar ratio of 2.4:1 approximately, with a molecular weight of 146 KDa. FT-IR, NMR and XRD spectra indicated that BSP faction contained α-Man and β-Glc residues with low overall crystallinity. The polysaccharide exhibited significant scavenging activities of ABTS and FRAP, as well as non-toxicity against human gastric epithelial GES-1 cells. Oral administration with 100 mg/kg of BSP for 3 days continuously could significantly prevent the formation of ethanol-induced gastric mucosal lesion. It could also reduce the levels of pro-inflammatory cytokines, including TNF-α, IL-1β, IL-6, and IL-18, and MPO activity in gastric tissue. Additionally, the BSP faction exhibited antioxidant activity, increased the content of PEG2 as a defensive factor, and suppressed MAPK/NF-κB signaling pathway in gastric tissue. These results indicated that the gastroprotective activity of BSP faction could be attributed to the reduction of pro-inflammatory cytokines and oxidative stress and the inhibition of MAPK/NF-κB pathways. Our results provided substantial evidence that BSP could be a promising phytomedicine for gastric ulcer prevention.