Wheat plants (Triticum aestivum L.) were subjected to varying phosphorus (P) supply and canopy 13CO2 feeding to uncouple the plant and soil factors regulating carbon (C) allocation between the shoot and roots and in the P-enriched v. P-deficient soil zone. In a split-root system, transferring from 200/200 µM P (high/high) to high/nil P or nil/nil P for 7 days was associated with 18–30% increase in the ratio of root-to-total 13C, whereas 8–12% more 13C was retained in the shoot of plants grown under continuous high/high P. Although the C signal between the shoot and roots weakened at day 12, it closely correlated with root P acquisition at both days. In a non-split-root system, plants supplied with 200 μM P (high) for 7 and 12 days had a lower ratio of root-to-total 13C than plants with continuous 20 μM P (low). Preferential C allocation and increased P acquisition occurred before any measurable growth difference. Shoot P status had a greater influence than soil P supply on plant C allocation, and rapid C signalling between the shoot and roots might serve as an important component of plant response to heterogeneous P conditions.