The potential for ingestion of copper oxide nanomaterials (CuO NMs) is increasing due to their increased exploitation. Investigation of changes in gene expression allows toxicity to be detected at an early stage of NM exposure and can enable investigation of the mechanism of toxicity. Here, undifferentiated Caco-2 cells, differentiated Caco-2 cells, Caco-2/HT29-MTX (mucus secreting) and Caco-2/Raji B (M cell model) co-cultures were exposed to CuO NMs andcopper sulphate(CuSO4) in order to determine their impacts. Cellular responses were measured in terms of production ofreactive oxygen species(ROS), the gene expression of an antioxidant (haem oxygenase 1 (HMOX1)), the pro-inflammatory cytokine (interleukin 8 (IL8)), the metal binding (metallothionein 1A and 2A (MT1AandMT2A)) and the mucus secreting (mucin 2 (MUC2)), as well as HMOX-1 protein level. While CuSO4induced ROS production in cells, no such effect was observed for CuO NMs. However, these particles did induce an increase in the level of HMOX-1 protein and upregulation ofHMOX1,MT2A,IL8andMUC2genes in all cell models. In conclusion, the expression ofHMOX1,IL8and MT2A were responsive to CuO NMs at 4 to 12h post exposure when investigating the toxicity of NMs using intestinalin vitromodels. These findings can inform the selection of endpoints, timepoints and models when investigating NM toxicity to the intestinein vitroin the future.