Information on the bioavailability and -accessibility of subsoil phosphorus (P) and how soil moisture affects its utilization by plants is scarce. The current study examined whether and to which degree wheat acquires P from subsoil allocated hydroxyapatite and how this could be affected by soil moisture. We investigated the 33P uptake by growing wheat in two rhizotron trials (soil and sand) with integrated 33P-labelled hydroxyapatite hotspots over a period of 44 days using digital autoradiography imaging and liquid scintillation counting. We applied two irrigation scenarios, mimicking either rainfall via topsoil watering or subsoil water storage. The plants showed similar biomass development when grown in soil, but a reduced growth in sand rhizotrons. Total plant P(tot) stocks were significantly larger in plants grown under improved subsoil moisture supply, further evidenced by enhanced P stocks in the ears of wheat in the sand treatment due to an earlier grain filling. This P uptake is accompanied by larger 33P signals, indicating that the plants accessed the hydroxyapatite because subsoil irrigation also promoted root proliferation within and around the hotspots. We conclude that even within a single season plants access subsoil mineral P sources, and this process is influenced by water management.