Acidic subsoils can restrict root growth and nitrogen (N) uptake and increase the risk of N loss; however, the extent of genotypic variation remains unclear. We evaluated two bread wheat cultivars, Haruyokoi and Harukirari, and one spelt line, KU-1025, under limed and acidic subsoil treatments to clarify whether maintaining root growth in acidic subsoil contributes to greater N capture and lower N loss. After 78 days, we measured shoot dry weight, shoot N uptake, root dry weight, total root length, soil nitrate (NO₃-N) concentration, and cumulative N leaching. The acidic subsoil reduced shoot N uptake, root length in the subsoil, deep-root ratio, and NO₃-N depletion, indicating that it restricted root proliferation and N acquisition. KU-1025 showed the greatest shoot dry weight, shoot N uptake, root dry weight, and total root length under both treatments. It also maintained a high deep-root ratio under acidic subsoil conditions and showed lower soil NO₃-N concentrations and less N leaching than the two wheat cultivars. Across genotypes and treatments, shoot N uptake was positively correlated with root dry weight and total root length, whereas N leaching was negatively correlated with these traits. These results suggest that maintaining a large root system, rather than deep rooting alone, is important for improving N capture and reducing N loss under acidic subsoil conditions, and that KU-1025 may provide useful genetic variation for breeding wheat adapted to acidic subsoil environments.