The Ryukyu long-furred rat, Diplothrix legata (Muridae) is an endangered large arboreal species endemic to the central Ryukyus, Japan. Previous studies have recorded the food habits of this species through direct observations. However, its observation records are limited. Here, we aimed to clarify the food habits of D. legata using DNA metabarcoding of its stomach contents. The ITS2 intergenic regions in plant nuclear genomes and the COI gene regions in animal mitochondrial genomes were used as DNA markers to identify the prey species of D. legata based on its stomach contents. We successfully identified 63 plant and 36 animal species as the prey species of D. legata. Finally, 84 plant and 46 animal species are listed as food menu in total. Thus, a greater number of its animal prey species were identified in this study than in previous observation-based studies. As this species is omnivorous, the conservation of a wide array of ecological habitats is necessary for maintaining its population. Regardless of the limited sample size and unknown parts consumed, slight differences in food items were observed between different sex- and age-based groups. Relatively more detailed DNA reference databases for the local fauna and flora are required for further analysis.