Subsurface acidity is a major factor limiting crop yield in some agricultural soils. The surface application of lime has limited effect on the subsurface acidity due to the slow downward movement, while deep incorporation of lime is costly. This paper tested the concept of biologically ameliorating subsurface acidity in a highly acidic soil through the net uptake of anions by plant roots. Nitrogen was supplied to the top soil (0–10 cm) as Ca(NO3)2 at rates equivalent to 30–240 kg N ha−1. Four water levels were imposed (40, 60, 80 and 100% of field capacity). Aluminium-tolerant wheat was grown for 58 days. The high N and high water treatments stimulated root growth below 15 cm, which in turn increased N capture, resulting in a greater excess anion uptake over cations and thus alkalisation of subsurface soil layers. This study suggests that it is feasible to exploit the process of nitrate uptake by an aluminium-tolerant wheat genotype to increase pH in acidic subsoil.