Aims: Arbuscular mycorrhizal fungi play important roles in plant phosphorus (P) accumulation. The aim of this study was to uncover how and to what extent soil plant-available P levels and maize genotypes influence the contribution of mycorrhizal P uptake pathway to plant P nutrition. Methods: We selected an old genotype HMY and a modern genotype XY335, combined with 32P labeling and qPCR to quantify P uptake efficiency of the direct pathway (DP) and the mycorrhizal pathway (MP) at three Olsen-P levels: 4.5 (low), 8 (medium) and 50 (high) mg kg−1. Results: The P uptake efficiency ratio PAE-MP/PAE-DP was highest in the treatment with medium Olsen-P, and was correlated positively with MP contribution. The traits of arbuscular mycorrhizal fungi, such as percent colonization, hyphal length density, P uptake per unit hyphae length, and the expression of the mycorrhiza-specific P transporter ZmPT1;6 were higher in XY335 than HMY in high-P soil, which was in accordance with the importance of the MP contribution. Conclusions: Greater mycorrhizal responsiveness in the modern maize genotype than the old genotype under high P soil condition is related to higher P uptake efficiency of MP than DP; the inherent potential of MP can be maximized by managing soil plant P availability to achieve optimal P supply in intensive farming.