The magnetite nanoparticles are progressively used in a wide range of biological applications. In the present study, we purposed to show apoptosis-inducing ability of Fe3O4 nanopowders on A549 cells. In addition, the toxic effects of Fe3O4 nanopowders were researched on L929 cells. The cytotoxicity of Fe3O4 nanopowders were evaluated on A549 and L929 cells by MTT assay and inhibited cell proliferation by time and dose-dependent manner on A549 cells but was not toxic on L929 cells. According to these findings, IC30 value of Fe3O4 nanopowders was determined as 5 µM. The early and late apoptotic cells were detected by Annexin V-FITC/PI assay using IC30 concentration of Fe3O4 nanopowders. Furthermore, The IC30 value of Fe3O4 nanopowders was not effective in the activation of caspase-3 but was effective on loss of mitochondrial membrane potential. The apoptotic index of A549 cells was investigated and found out to increase by IC30 value of Fe3O4 nanopowders using TUNEL, BrdU, Bcl-2 immunocytochemical assays. The upregulated and downregulated genes were profiled and the presence of some apoptotic genes was determined with administration of IC30 value of Fe3O4 nanopowders by microarray assay. This work suggests that Fe3O4 nanopowders could be a good candidate for therapy of lung adenocarcinoma cells.