Analyzing variations in silicon (Si) isotopes can help elucidate the biogeochemical Si cycle and Si accumulation processes of higher plants. Importantly, the composition of Si isotopes in higher plants has yet to be studied comprehensively and our knowledge of the distribution of Si isotopes in higher plants lags behind that of Si isotopes in marine organisms, such as diatoms. In the present study, we investigated the isotope fractionation that occurs during the uptake and transport of Si in rice, using a series of hydroponic experiments with different external concentrations of Si. We found that an active mechanism was responsible for the majority of Si uptake and transport at lower Si levels and that the uptake of Si by rice roots was significantly suppressed by both low temperature and metabolic inhibitors. In addition, light Si isotopes (28Si) entered roots more readily than heavy Si isotopes (30Si) when the active mechanism was inhibited. Therefore, we conclude that biologically mediated isotope fractionation occurs during the uptake of Si by rice roots. In addition, both active and passive Si uptake components co-exist in rice, and the fractionation effect is enhanced when more Si is absorbed by plants.