Despite the great interest in nanoparticles (NPs) safety, no comprehensive test paradigm has been developed. Oxidative stress has been implicated as an explanation behind the toxicity of NPs. It is reported that sulphoraphane (SFN) present in cruciferous vegetables like cauliflower and broccoli has potential to protect cells from oxidative damage and inflammation. However, protective role of SFN in nanotoxicity is not explored. We investigated the protective effect of SFN against the toxic response of copper oxide (CuO) NPs in mouse embryonic fibroblasts (BALB 3T3). Results showed that CuO NPs induced dose-dependent (5-15 µg/ml) cytotoxicity in BALB 3T3 cells demonstrated by MTT and lactate dehydrogenase (LDH) assays. CuO NPs were also found to induce oxidative stress in dose-dependent manner indicated by induction of reactive oxygen species (ROS) and lipid peroxidation (LPO) and depletion of glutathione and glutathione reductase. Co-treatment of BALB 3T3 cells with SFN (6 µM) significantly attenuated the cytotoxicity, ROS generation and oxidative stress caused by CuO NPs. Moreover, we found that co-treatment of another antioxidant N-acetyl-cysteine (NAC) (2 mM) also significantly attenuated glutathione depletion caused by CuO NPs but protection from the loss of cell viability due to CuO NPs exposure was not significant. We believe this is the first report showing that SFN significantly protected the BALB 3T3 cells from CuO NPs toxicity, which is mediated through generation of oxidants and depletion of antioxidants. Consequently, protective mechanism of SFN against CuO NPs toxicity was different from NAC that should be further investigated.