The Iheya North Knoll in the mid-Okinawa Trough is a methane-rich deep-sea hydrothermal field. To cultivate methane-oxidizing bacteria from the Iheya North field, a continuous flow-through reactor cultivation was conducted using gill tissue of Bathymodiolus japonicus as an inoculum. The enriched culture consisting mainly of methane-oxidizing bacteria was subjected to genetic analyses to investigate their metabolic characteristics.