Global soil carbon (C) stocks account for approximately three times that found in the atmosphere. In the Aso mountain region of Southern Japan, seminatural grasslands have been maintained by annual harvests and/or burning for more than 1000 years. Quantification of soil C stocks and C sequestration rates in Aso mountain ecosystem is needed to make well-informed, land-use decisions to maximize C sinks while minimizing C emissions. Soil cores were collected from six sites within 200 km2 (767–937 m asl.) from the surface down to the k-Ah layer established 7300 years ago by a volcanic eruption. The biological sources of the C stored in the Aso mountain ecosystem were investigated by combining C content at a number of sampling depths with age (using 14C dating) and δ13C isotopic fractionation. Quantification of plant phytoliths at several depths was used to make basic reconstructions of past vegetation and was linked with C-sequestration rates. The mean total C stock of all six sites was 232 Mg C ha−1 (28–417 Mg C ha−1), which equates to a soil C sequestration rate of 32 kg C ha−1 yr−1 over 7300 years. Mean soil C sequestration rates over 34, 50 and 100 years were estimated by an equation regressing soil C sequestration rate against soil C accumulation interval, which was modeled to be 618, 483 and 332 kg C ha−1 yr−1, respectively. Such data allows for a deeper understanding in how much C could be sequestered in Miscanthus grasslands at different time scales. In Aso, tribe Andropogoneae (especially Miscanthus and Schizoachyrium genera) and tribe Paniceae contributed between 64% and 100% of soil C based on δ13C abundance. We conclude that the seminatural, C4-dominated grassland system serves as an important C sink, and worthy of future conservation.