Abstract: With extensive application of Fe₃O₄ nanoparticles in a variety of fields, the possibility of their exposure to agricultural environment is growing. This raises the concerns on their effects on environment. In this investigation, we carried out a pot experiment to investigate the impacts of diffe-rent concentrations of Fe₃O₄ nanoparticles (1, 10, 100 mg·kg^-1), as well as their counterparts, bulk Fe₃O₄ particles, on the lettuce growth and the soil bacterial community. We not only measured the net photosynthetic rate of plant leaves, the Fe content in plant, but also determined the soil bacterial community structure and components using the high throughput technique. Results showed that responses of the plant and bacterial community varied with the increase of Fe₃O₄ nanoparticles concentration. At the low concentration, Fe₃O₄ nanoparticles stimulated plant growth, accompanied by the increased abundance of Xanthomonadales and the decreased abundance of Cyanobacteria and Sphingobacteria, but no significant changes were observed for the bacterial alpha diversity indices. At the high concentration, Fe₃O₄ nanoparticles could not only suppress plant growth, increase Fe content in plant tissue and soil electronic conductivity, but also decrease the phylogenetic diversity of bacterial community, reduce the abundances of Xanthomonadales and Sphingobacteria, and increase the proportion of Cyanobacteria. Moreover, the responses of some functional guilds in soil bacterial community varied between the Fe₃O₄ nanoparticle treatment and the bulk counterpart. This indicated that the size and the concentration of Fe₃O₄ were the factors influencing soil bacterial community, which would potentially impact the plant development. Therefore, more attention should be focused on soil microbes when evaluating the biological effects of nanoparticles.