How plants balance different strategies to acquire resources from the environment is a basic question in biology and ecology. Phosphorus (P) uptake by mycorrhizal plants comprises direct pathway (DP) and mycorrhizal pathway (MP). It is unknown whether there is a trade-off between DP and MP and how plants govern selection and/or differential contributions of the two pathways. We used 32P labelling technique to quantify P uptake by both pathways in three maize genotypes differing in P acquisition at Olsen P values of 4.5, 8 and 50 mg kg−1, representing low, suboptimal and high P supplies, respectively. We calculated the preference index of P uptake (ratio of MP contribution to DP contribution), P-transporter-based preferential index, C investment index as (ratio of hyphal length to root length), and the P-efficiency-based preferential index. Our results showed that maize plants preferred MP at the suboptimal soil available P because both the C investment index and the P-efficiency-based preferential index were greatest at that P supply. Shoot P content showed a significant relationship with the P uptake preference index, suggesting shoot P demand governed the trade-off between DP and MP. In conclusion, the preference for DP or MP regarding P acquisition in maize shifts with changes in soil available P level. These findings could be harnessed to better manage mycorrhizal fungi to enhance the sustainability of agricultural crops in soils with suboptimal P availability.