With the increasing application of Fe3O4 nanoparticles, it is necessary to investigate their safety to human cells. It has been revealed in this study that the colony-forming ability of HeLa cells is not influenced by Fe3O4 nanoparticles, indicating that Fe3O4 nanoparticles do not exhibit cytotoxicity to those cells. However, Fe3O4 nanoparticles significantly inhibit the colony-forming ability of XPF cells, which contain mutated XPF gene and possess a deficient nucleotide excision repair (NER) pathway, with a concentration dependent pattern. Our data illustrate that Fe3O4 nanoparticles are cytotoxic to XPF cells. HeLa cells and XPF cells were incubated with Fe3O4 nanoparticles containing cell culture media. The mRNA level of several genes, which were involved in cell cycle regulation or the cellular response to stress, has been detected by RT-PCR assay. Only significant elevated mRNA level of CHEK1, RPA1, RPA2 and RPA3 has been observed in XPF cells. The results imply that Fe3O4 nanoparticles are not suitable for patients with any mutated NER gene.