Toxicogenomics is a developing area searching for cellular pathways and mechanisms including cancer, immunological diseases, environmental responses, gene-gene interactions and drug toxicity. Nanoparticles (NPs) become important candidates for analyzing in toxicogenomic experiments because of their unusual properties in various biological activities. Therefore, we examined the nanotoxicity of iron oxide (Fe2O3) on gene expression profiling of human alveolar epithelial cells (HPAEpiC) in the study. For this aim, iron oxide nanoparticles were synthesized by zone melting method and characterized via using X-ray crystallography (XRD) and transmission electron microscope (TEM) techniques. Cell viability and cytotoxicity were determined by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) release tests. Whole-genome microarray expression analysis was performed to explore the effects of iron oxide nanoparticles on gene expression in cultured human alveolar epithelial cells. For further analyses, these genes were functionally classified by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) with gene ontology (GO) analysis. The results from this study indicated that iron oxide-mediated toxicity directly or indirectly affecting the regulation of cell proliferation, response to hormone stimulus, estrogen stimulus, cytokine activity and blood circulation by stimulating diverse genes.