Plants typically take up Fe through either strategy I or strategy II, whereas rice uses both. Stable Fe isotopes potentially reveal pathways of Fe uptake in rice plants. In this study we measured Fe isotopic compositions of rice grown under different conditions, i.e. paddy soil with deficient Fe supply and Fe³⁺-EDTA aqueous solution with sufficient Fe supply, to investigate whether Fe isotope fractionation is distinct under Fe-deficient and Fe-sufficient conditions as well as their possible controlling mechanisms. Our results show that rice grown in the Fe³⁺-EDTA aqueous solutions with sufficient Fe supply preferentially takes up light Fe isotopes (Δ⁵⁶Fe_{bulk plant-nutrient} = −1.36‰) and accumulates Fe in different parts of plant (roots, stems, leaves, husks, and grains) with large Fe isotope fractionation. Under such a growing condition, rice takes up Fe through strategy I and the reduction of Fe³⁺ to Fe²⁺ results in plants enriching isotopically light Fe. Within the plant, the transportation of Fe is accompanied with changes in redox state, which thus causes significant Fe isotope fractionation among different plant tissues. In contrast, rice plants grown in soils with deficient Fe supply are slightly enriched in heavy Fe isotopes (Δ⁵⁶Fe_{bulk plant-soil solution} = 0.27‰) and accumulate Fe in different plant tissues with little isotope fractionation. Under this growing condition, the rice plant takes up Fe through strategy II. Plants take up Fe from soils as Fe³⁺-phytosiderophores (Fe³⁺-PS) complex and transport Fe as Fe³⁺-nicotianamine (Fe³⁺-NA) complex throughout the plant, which does not involve changes in redox state and thus results in very limited isotope fractionation. The observed difference in Fe isotope fractionation indicates two distinct Fe uptake and translocation strategies for rice grown under the two different conditions: strategy I under Fe-sufficient conditions, and strategy II under Fe-deficient conditions. Our results demonstrate that Fe isotope ratios may be used to distinguish Fe-sufficient versus Fe-deficient conditions and to elucidate Fe biogeochemical processes during rice growth.