Nine human remains were recovered from Shiraho-Saonetabaru Cave on Ishigaki Island, Okinawa Prefecture, Japan, between 2007 and 2009. Six of the nine samples produced well-preserved biogenic collagen, which was submitted to radiocarbon dating by accelerator mass spectrometry. Three human samples (Nos. 2, 4, and 8) from the fossil chamber were dated to between 16 and 20 ka BP, and can clearly be assigned to the Late Pleistocene. One animal bone from the same chamber which was treated and measured for radiocarbon independently was also of great antiquity (c. 12 ka BP). These dates are the first concrete evidence of human occupation on the Ryukyu Islands during the Pleistocene, based on the direct radiocarbon dates of human remains. It is expected that more human remains and archaeological objects of the Pleistocene will be recovered from Shiraho-Saonetabaru Cave and the surrounding region by future intensive collaborations between anthropologists, archaeologists, and speleologists.