【Objective】The low availability of iron in calcareous soil leads to serious iron-deficiency chlorosis in plants. Thus, it is important to screen highly efficient iron-solubilizing bacteria and explore their collaboration with mycorrhizal fungi (AM) to increase the available iron content and improve plant iron nutrition. 【Method】Pot experiments were conducted with calcareous soil and tomato (Lycopersicon esculentum) as test materials. They were inoculated with Advenella kashmirensis (B1), Arthrobacter cupressi (B2), Klebsiella variicola (B3), Variovorax guangxiensis (B4) and Enterobacter ludwigii (B5), and treatment with no bacteria inoculation as the control group (CK). Efficient iron-solubilizing bacterial strains B1, B2 and B3 were screened and combined with AMF (Rhizoshagu irregularis, Ri) as B1+Ri, B2+Ri, B3+Ri and B1+B2+B3+Ri treatments. Also, AM fungi were inoculated alone (Ri) to explore the mechanism of synergistic effect between different iron-solubilizing bacteria and AMF to mobilize insoluble iron in calcareous soil and promote iron absorption in plants. 【Result】The results showed that compared with the control treatment, inoculation with B1, B2 and B3 strains could significantly increase the tomato biomass and the total iron accumulation in root and shoot increased by 6.48 and 2.61, 4.11 and 2.03, 4.37 and 2.25 times, respectively. The active iron content in new leaves increased by 74.21%, 133.66% and 175.84%. Compared with inoculation with AMF alone, different co-inoculation combinations significantly increased the tomato biomass, and the average total iron accumulation in different parts of the plant increased by 58.32%-119.43%. Under B3+Ri and B1+B2+B3+Ri treatments, the active iron content in tomato roots increased by 41.47% and 44.30%, and new leaves increased by 12.61% and 12.77%, respectively. Different co-inoculation combinations could effectively improve the root architecture of the plant, and the mycorrhizal infection rates of AM fungi under different co-inoculation treatments were 13.35%-30.99% higher than those under inoculation alone. The root iron reductase activity was significantly increased by 9.86%-22.07% compared with the inoculation with AM fungi alone, and the relative expressions level of LeFIT1, LeFRO2 and LeMYB72 in tomato roots was significantly up-regulated. Compared with exclusive AMF inoculation, B3+Ri and B1+B2+B3+Ri treatments reduced the rhizosphere soil pH value by 0.21 and 0.09, respectively, but increased the soil available Fe content by 15.78% and 55.23%. 【Conclusion】It was concluded that the synergistic effect of AM fungi and three high-efficiency iron-solubilizing bacteria could significantly improve iron availability in calcareous soil and enhance plant iron nutrition. However, the synergistic mechanism between different types of iron-solubilizing bacteria and AMF was different and provided a microbial approach to solve the problem of low iron availability in calcareous soil.