We assessed the dietary profile of the large Japanese wood mouse (Apodemus speciosus) inhabiting the islands of the Seto Inland Sea and adjacent areas by DNA metabarcoding analyses of feces with the chloroplast trnL (UAA) P6 loop intron region and the mitochondrial cytochrome c oxidase subunit I gene as DNA-barcoding markers. The results indicated that A. speciosus consumed a variety of food items. Fagaceae (Quercus sp.) and Rosaceae were frequently utilized plant families, and the Lepidoptera (Noctuidae) was the frequently utilized animal family. The acorn-producing Quercus species was found to be a fundamental diet, while Rosaceae might be characteristic in temperate regions. The high dependence on the Noctuidae moths as an animal dietary component implies that A. speciosus may be reducing the pest abundance in orchards. Technical pitfalls surrounding the DNA metabarcoding methods for feces of the small rodent were also discussed.