We investigated the protective effect of a hydrous ethanol extract of brown rice fermented with Aspergillus oryzae (ERF) which contained nucleobases and low fiber on the methotrexate (MTX)-induced gastrointestinal damage in rats. The rats were assigned to three groups: control (CON), MTX, and MTX-ERF. The rats in the CON and MTX groups were fed for 4 weeks on a basal diet, and those in the MTX-ERF group were fed on a 9.16% ERF-containing basal diet. The rats in the MTX and MTX-ERF groups were administered with MTX after 3 weeks. The survival rate and incidence rate of diarrhea were monitored over 1 week. On day 4 after the administration, half of the rats in each group were killed, and gastrointestinal samples were collected. Feeding with ERF improved the incidence rate of diarrhea, increased the protein content in small intestinal mucosa, and also apparently improved the survival rate. These results indicate that dietary ERF could protect against MTX-induced gastrointestinal damage.