Fe3O4 magnetic nanoparticles (Fe3O4 MNPs) have attracted increasing attention in recent years due to their extensive applications. However, their effects on cells and relevant molecular mechanisms remain not very clear. In this paper, the effects of Fe3O4 MNPs on the proliferation and apoptosis of human HeLa cells, the activation of transcription factor NF-kappaB and the expressions of its target genes in HeLa cells were investigated. The entrance of nanoparticles into cells was demonstrated by Prussian blue staining and transmission electron microscopy (TEM) and it revealed that the majority of nanoparticles existed in cytoplasm in a collective format. The amount of nanoparticles in cells was dependent on the dose and incubating time acting to cells. It was revealed that Fe3O4 MNPs exerted no significant effects on the proliferation of HeLa cells and did not induce apoptosis of HeLa cells. The activation of NF-kappaB and the expressions of its typical target genes, IL-8, COX-2 and MCP-1, were not significantly changed under the various concentrations of Fe3O4 MNPs. Therefore, it is concluded that the Fe3O4 MNPs have a good biocompatibility to HeLa cells cultured in vitro and do not affect NF-kappaB signaling pathway under the dose and acting time used in this study.