We investigated the impact of predatory crabs and fishes on hatchery-reared juvenile Japanese flounder Paralichthys olivaceus released into a flounder nursery habitat in Mano Bay, Sado Island. In 2001 and 2002, nearly 50,000 reared juveniles (ca. 6.1 cm total length, TL) were released each year, and 68% and 83% of them were estimated to suffer mortality in the first week after release, respectively, for these years. Stomach content analysis of crabs caught in the released area, using a PCR-based technique for detecting Japanese flounder-specific mtDNA, revealed that the swimming crab Charybdis japonica is a significant predator on released flounder. This finding was supported by laboratory observations that the crabs readily preyed on live reared flounder. In addition, through microscopic observations of stomach contents of piscivorous fishes, four species (devil stinger Inimicus japonicus, age-1 Japanese flounder, spotted flathead Cociella crocodila, and bartailed flathead Platycephalus indicus) were identified as predators. Based on stomach content analyses and abundances of these predators, consumption by swimming crabs is estimated to account for 39% and 41% of the loss of released flounder during the first week after release in 2001 and 2002, respectively, whereas consumption by four piscivorous fishes accounted for 25% and 20% of the loss in 2001 and 2002, respectively. Thus, we conclude that predation by crabs and fishes is the major cause of mortality of released flounder shortly after release.