Objective Long-term excessive phosphorus (P) application in intensive agro-ecosystem leads to P accumulation in soil. Whether there was a legacy effect on accumulated P in soil.Method This study was based on a long-term experiment (started in 2007) in North China Plain. The microorganisms with different P supply levels (from P₂O₅ 0, 75, 300 kg·hm^–2, P0, P75, P300, respectively) in calcareous soil was selected as the research object. Pot microbial inoculation experiment was conducted to explore the soil microorganisms training with gradient P fertilization on plant growth, nutrition absorption, soil enzyme activities and mycorrhizal characteristics under two substrate P supply levels (0 mg·kg^–1, 30 mg·kg^–1, no P, + P, respectively).Result The type of inoculants (original inoculants, sterilization treatment) significantly affected the aboveground biomass and phosphorus uptake of plants. Inoculating original inoculants (P0, P75 and P300) significantly increased the aboveground biomass and P uptake of clover compared with sterilized inoculants under the condition of two substrate P levels, but there was no significant difference among the three original inoculants. The aboveground biomass of maize inoculated with original inoculants P300 was significantly higher than that in the treatments of P0 and P75. At the same time, the inoculant and substrate P supply level co-affected soil enzyme activities. Compared with P0 and P75 inoculants, P300 inoculant significantly decreased the peroxidase activities in the soil of clover under no P and + P conditions, and significantly increased the peroxidase and chitinase activities in the soil of maize under no P conditions. Also, compared with inoculating in original P0 and P300 inoculants, P75 inoculant significantly increased the activities of acid and alkaline phosphatase in maize soil under no P condition. In addition, the mycorrhizal colonization differed due to plant species. Compared with P0 and P75 inoculants, the colonization rate of arbuscular mycorrhizal fungi in clover root significantly reduced after inoculating with P300, but there was no significant difference among the three original inoculants in maize root.Conclusion The substrate P supply and plant species co-affect microorganisms training by accumulated P, suggesting that plant-microbial characteristics need to be considered in P fertilizer management.