The experiment was carried out with subcutaneous injections of Methotrexate (MTX), a commonly used antimetabolite drug, is known to induce intestinal inflam- mation. We investigated whether a hot-water extract of Prasiola japonica Yatabe (PJ) can be used as an effective remedy against MTX induced intestinal injury. A hot-water extract of PJ was administered orally to mice with MTX-induced intestinal inflammation. Experiments were carried out in which the mice were given PJ by gavage at an initial dose of 5 or 10 mg/kg body weight for 6 days. The mice were also given a subcutaneous injection of MTX (7.0 mg/kg body wt/day) daily throughout the 6-day experimental period. The shortening of the small intestine and gastrointestinal mucosal ulceration induced by MTX were attenuated following oral administration of the PJ extract. The numbers of apoptotic cells and Proliferating Cell Nuclear Antigen-positive cells in the intestinal mucosa of the PJ-treated mice were lower than those in the MTX- treated mice. These results indicate that the extract of PJ could protect against MTX- induced damage of the small intestine. Folic acid was found to be present in the PJ extract at the concentration of about 2.5μg/mg, and may be considered as having been responsible for the decrease in the severity of the MTX-induced damage of the small intestine.