A new species of the genus Callobius is described from Amami-Ōshima Island, southwestern Japan, under the name of Callobius amamiensis n. sp., and the differences from the related species, C. yakushimensis Okumura, 2010 are shown. The male palp of the new species is characterized by having the short mesal process in the tibia. In the female specimen, the lateral lobes of the epigyne are situated distant from each other, hence the width of epigyne is almost twice the length. In addition, the mitochondrial cytochrome oxidase subunit I (mt-COI) partial sequences of the new species have been analyzed and documented for future use.