Chemical and isotopic compositions of hydrothermal fluids from Hatoma Knoll in the southern Okinawa Trough were investigated. The hydrothermal fluids were derived from a single pure hydrothermal fluid source, but they underwent phase separation beneath the seafloor prior to venting. Only vapor-like fluids vent at the Hatoma system, and the most active area is around the center of the crater, based on the location of the maximum temperature and the lowest Cl- concentrations. Compared with other hydrothermal systems in the world, at Hatoma the pH and alkalinity, as well as the B, NH4+, K, Li, CO2, and CH4 concentrations, were higher, and the Fe and Al concentrations were lower, suggesting that the characteristics of the Hatoma hydrothermal fluids are comparable to those of the other Okinawa Trough hydrothermal fluids. Helium isotope ratios were lower than those of sediment-starved hydrothermal systems, suggesting that He-4 derived from the sediment is supplied to the hydrothermal fluids in Hatoma Knoll. The carbon isotope ratios of CO2 in the hydrothermal fluids indicate an influence of organic carbon decomposition. The carbon isotope ratios of CH4 in the hydrothermal fluids imply that most of the CH4 originated from microbial methane produced in a recharge zone of the hydrothermal system. Although sediment influences are a typical feature of Okinawan Trough hydrothermal fluids, the Hatoma hydrothermal system has the lowest carbon isotope ratios of CH4 among them, which suggests that Hatoma is the most highly influenced by the sediments in the recharge zone. Thus, the degree of the sediment influences has a variable in each hydrothermal field in the Okinawa Trough.