Angiostrongylus cantonensis is a zoonotic nematode with rodents serving as natural definitive hosts. We report A. cantonensis in the Ryukyu Islands tree rat (Diplothrix legata, Thomas, 1906), a native endangered species in Japan. Adult and larvae of A. cantonensis were macroscopically, histologically, and genetically detected in three tree rats collected between August 2011 and January 2012 in the Yambaru area of Okinawa Prefecture, Japan. Pathologic observations of the lungs of rats showed that infection may be lethal. We also conducted a retrospective genetic survey of helminths parasitic in lung in cryopreserved lung samples of Ryukyu Islands tree rats collected between 2007 and 2011 in the Yambaru area and found A. cantonensis DNA in one of 29 samples, which was collected in December 2010.