The aim of this study was to evaluate the effectiveness of nitrilotriacetic acid (NTA) on cadmium (Cd) fractions and microbial biomass in a calcareous soil spiked with Cd under cultivated (Zeamays L.) and uncultivated regime subject to soil leaching condition. Expanding investigations related to soil-plant interactions on metal-contaminated soils with insights on microbial activity and associated soil toxicity perspective provides novel perspectives on using metal-chelating agents for soil remediation. The experimental factors were three levels of Cd contamination (0, 25, and 50mgkg-1 soil) and three levels of NTA (0, 15, and 30mmol L-1) in loamy soil under maize-cultured and non-cultured conditions. During the experiment, the adding NTA and leaching processes were performed three times. The results showed that the amount of leached Cd decreased in cultivated soil compared to uncultivated soil due to partial uptake of soluble Cd by plant roots and changes in Cd fractions in soil, so that Cd leached in Cd50NTA30 was 9.2 and 6.1mg L-1, respectively, in uncultivated and cultivated soils. Also, Cd leached in Cd25NTA30 was 5.7 and 3.1mg L-1 respectively, in uncultivated and cultivated soils. The best treatment in terms of chemical and microbial characteristics of the soil with the high percentage of Cd removed from the soil was Cd25NTA30 in cultivated soil. In Cd25NTA30 compared to Cd25NTA0 in cultivated soil, pH (0.25 unit), microbial biomass carbon (MBC, 65.0mgkg-1), and soil respiration (27.5mg C-CO2 kg-1 24h-1) decreased, while metabolic quotient (qCO2, 0.05) and dissolved organic carbon (DOC, 20.0mg L-1) increased. Moreover, the changes of Cd fractions in Cd25NTA30 in cultivated soil compared to uncultivated soil were as follows; the exchangeable Cd (F1, 0.27mgkg-1) and Fe/Mn-oxide-bounded Cd (F4, 0.15mgkg-1) fractions increased, in contrast, carbonate-Cd (F2, 2.67mgkg-1) and, organically bounded Cd (F3, 0.06mgkg-1) fractions decreased. NTA had no significant effect on the residual fraction (F5). The use of NTA, especially in calcareous soils, where most of the Cd is bound to calcium carbonate, was able to successfully convert insoluble fractions of Cd into soluble forms and increase the removal efficiency of Cd in the phytoremediation method. NTA is a non-toxic chelating agent to improve the accumulation of Cd in maize.