Recently, attempts have been made to apply graphene oxide (GO) in the field of biology and medicine, such as DNA sensing and drug delivery with some necessary modifications. Therefore, the toxicity of GO must be evaluated before it is applied further in biomedicine. In this paper, the cytotoxicity and genotoxicity of GO to human lung fibroblast (HLF) cells have been assessed with methyl thiazolyl tetrazolium (MTT), sub-G1 measurement and comet assays, and the mechanism of its toxicity has been explored. Various modifications of GO have been made to help us determine the factors which could affect the toxicity of GO. The results indicated that cytotoxicity and genotoxicity of GO to HLF cells were concentration dependent, and the genotoxicity induced by GO was more severe than the cytotoxicity to HLF cells. Oxidative stress mediated by GO might explain the reason of its toxic effect. Furthermore, the electronic charge on the surface of GO would play a very important role in the toxicity of GO to HLF cells. Copyright © 2013 John Wiley & Sons, Ltd.