Beyond its role in the calcium and phosphate homeostasis, calcitriol – the physiologically active form of 1,25 dihydroxyvitamin D3 – is known as an important modulator of cellular proliferation, differentiation, inflammation, and immune systems.(1) The aim of the present study was to determine if vitamin D3 has a protective effect against tissue injury in a rat model of HCl/ethanol-induced gastric ulcer. Sprague-Dawley rats of both sexes (250-300 g; n=8 per group) were fasted for 24 hours. A gastric injury was induced by acidified ethanol solution (0.3 M HCl/60% ethanol) per os (0.2 ml). Control group received saline (0.2 ml, per os). Ulcer groups were treated with 1,25 dihydroxyvitamin D3 (VitD; 0.25 mg/kg; intraperitoneally) for 14 days alone or along with the non-selective nitric oxide synthase inhibitor L-NAME (20 mg/kg; intraperitoneally), the inhibitor of sulfhydryl groups N-ethylmaleimide (NEM; 10 mg/kg; intraperitoneally), ATP-sensitive K+ channel blocker glibenclamide (10 mg/kg; orally) or the non-selective cyclooxygenase inhibitor indomethacin (Indo; 10 mg/kg; subcutaneously). On day-15, the rats underwent ulcer induction by HCl/ethanol and decapitated 60 min later. The stomachs were examined macroscopically. Stomachs and trunk blood were sampled for biochemical assays. All procedures were approved by the Marmara University, Animal Care and Use Committee. Values are means±SEM, compared using ANOVA and Student’s t-tests. Gastric macroscopic lesion score of the untreated ulcer group (33.13±5.09) was decreased by pretreatment with VitD (19.00±4.34; p<0.05) and this effect was augmented by L-NAME (0.11±0.05; p<0.01) and attenuated by Indo (45.33±6.04; p<0.01) given along with VitD. Ulcer group revealed increased gastric malondialdehyde (MDA) (15.20±1.64 nmol/g; p<0.001), reduced endogenous antioxidant glutathione (GSH) levels (0.69±0.09 mmol/g; p<0.05) and increased myeloperoxidase (MPO) activity (20.96± 0.86 U/g; p<0.001) in comparison to control. The changes in these parameters were effectively suppressed by VitD (7.63±0.31 nmol/g; p<0.01, 1.33±0.16 mmol/g; p<0.01 and 8.82±0.41; p<0.001 U/g, respectively). Indo reversed the effect of VitD on MDA, GSH and MPO levels (p<0.05-p<0.001) whilst NEM had a slighter effect on gastric MDA and MPO levels achieved by VitD (p<0.05-p<0.001). Gastric levels of the endogenous antioxidants superoxide dismutase (SOD) and catalase did not show statistically significant changes in the untreated ulcer group in comparison to control and VitD did not seem to change these parameters. In conclusion, pretreatment with VitD protected the gastric tissue against oxidant injury in a rat model of HCl/ethanol-induced gastric injury via mechanisms possibly involving the cyclooxygenase system.