The aim of this study was to investigate the effect of Lactobacillus plantarum HFY09 on gastric injury induced by HCl/ ethanol in Kunming mice. The results showed that HFY09-H inhibited any increases in gastric juice volume, maintained the normal pH value of gastric acid, and reduced the damage caused to the gastric mucosa and gastric wall, the inhibition rate on the injury area reaches 63.70%. Compared with the negative control group, HFY09 increased the levels of serum somatostatin (SS) and vasoactive intestinal peptide (VIP), and also decreased the levels of substance P (SP), endothelin-1 (ET-1), interleukin-6 (IL-6), interleukin-12 (IL-12), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). In addition, real time fluorescent quantitative PCR (Q-PCR) also confirmed that high-dose HFY09 (10 9 CFU/kg/ day) upregulated the mRNA expression of copper/zinc superoxide dismutase (Cu/Zn-SOD), manganese superoxide dismutase (Mn-SOD), catalase (CAT), endothelial nitric oxide synthase (eNOS), and neuronal nitric oxide synthase (nNOS), and downregulated the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). At the same time, the results of the HFY09 treatment group were similar to those of the ranitidine treatment group. These results indicate that HFY09 can prevent gastric injury induced by HCl/ethanol in vivo. Therefore, HFY09 may play a potential role in the treatment of gastric diseases.