1. Re-allocation of nutrients from roots to shoots is essential for plant regrowth in grasslands, particularly in nutrient-poor conditions. However, the response of root nutrient re-allocation to changes in nitrogen (N) and water availability remains largely unknown.

2. Using a novel ¹⁵N and ³²P labelling technique, we quantified the contribution of N and phosphorus (P) to shoot regrowth from either root re-allocation or direct soil uptake for perennial grasses exposed to high-frequency deficit irrigation (HFDI) and N addition.

3. Without N addition, HFDI showed no impact on uptake and re-allocation of N and P, likely due to unaffected soil N availability and a greater diffusion barrier offsetting increased accumulation in plant-available soil P. With N addition, HFDI increased plant N rather than P uptake, because of increasing soil N availability instead of P under combined HFDI and N addition. The HFDI decreased both N and P re-allocation with N addition, possibly due to exhaustion of nutrient reserves in roots that were re-allocated aboveground. Re-allocation contributed 48-97% of N and 58-79% of P required during the first two weeks of shoot regrowth.

4. Synthesis. Our results highlight the importance of N and P re-allocation from roots to buffer against changes in soil N and P availability and to maintain N:P ratio in shoot regrowth.