Gastric ulcers (GUs) are a common type of peptic ulcer. Alcohol overdose is one of the main causes of GU, which is difficult to prevent. Although the protective effect of butyrate on inflammation-related diseases is well understood, its effect on GUs has not been reported. We investigated the protective effects of butyrate against ethanol-induced lesions to the gastric mucosa in mice and the underlying mechanisms. BALB/c mice were orally pretreated with butyrate for 30min prior to the establishment of the GU model by challenge with absolute ethanol. Ethanol administration produced apparent mucosal injuries with morphological and histological damage, whereas butyrate pretreatment reduced the gastric mucosal injuries in a dose-dependent manner. Butyrate pretreatment also significantly ameliorated contents of malondialdehyde (MDA) and carbonyl proteins, and decreased levels of IL-1β, TNF-α and IL-6. The Western blot results consistently demonstrated that butyrate pretreatment attenuated the phosphorylation of NF-κB p65, p38 MAPK and ERKs in the gastric tissues. Additionally, gastric wall mucus (GWM), a parameter reflecting mucosal defense, was clearly increased by butyrate pretreatment. Butyrate pretreatment protects the gastric mucosa against ethanol-induced lesions by strengthening the mucosal defense and anti-oxidant and anti-inflammatory activities. As a necessary substance for the body, butyrate may be applied to the prevention and treatment of GUs.