The aim of the present study was to investigate the gastroprotective activity of a sulfated-polysaccharide (PLS) fraction extracted from the marine red algae Gracilaria caudata and the mechanism underlying the gastroprotective activity. Male Swiss mice were treated with PLS (3, 10, 30 and 90 mg·kg(-1), p.o.), and after 30 min, they were administered 50% ethanol (0.5 mL/25 g(-1), p.o.). One hour later, gastric damage was measured using a planimeter. Samples of the stomach tissue were also obtained for histopathological assessment and for assays of glutathione (GSH) and malondialdehyde (MDA). Other groups were pretreated with l-NAME (10 mg·kg(-1), i.p.), dl-propargylglycine (PAG, 50 mg·kg(-1), p.o.) or glibenclamide (5 mg·kg(-1), i.p.). After 1 h, PLS (30 mg·kg(-1), p.o.) was administered. After 30 min, ethanol 50% was administered (0.5 mL/25 g(-1), p.o.), followed by sacrifice after 60 min. PLS prevented-ethanol-induced macroscopic and microscopic gastric injury in a dose-dependent manner. However, treatment with l-NAME or glibenclamide reversed this gastroprotective effect. Administration of propargylglycine did not influence the effect of PLS. Our results suggest that PLS has a protective effect against ethanol-induced gastric damage in mice via activation of the NO/K(ATP) pathway.