Copper oxide nanomaterials (CuO NMs) are exploited in a diverse array of products including antimicrobials, inks, cosmetics, textiles and food contact materials. There is therefore a need to assess the toxicity of CuO NMs to the gastrointestinal (GI) tract since exposure could occur via direct oral ingestion, mucocillary clearance (following inhalation) or hand to mouth contact. Undifferentiated Caco-2 intestinal cells were exposed to CuO NMs (10 nm) at concentrations ranging from 0.37 to 78.13 μg/cm Cu (equivalent to 1.95 to 250 μg/ml) and cell viability assessed 24 h post exposure using the alamar blue assay. The benchmark dose (BMD 20), determined using PROAST software, was identified as 4.44 μg/cm for CuO NMs, and 4.25 μg/cm for copper sulphate (CuSO ), which informed the selection of concentrations for further studies. The differentiation status of cells and the impact of CuO NMs and CuSO on the integrity of the differentiated Caco-2 cell monolayer were assessed by measurement of trans-epithelial electrical resistance (TEER), staining for Zonula occludens-1 (ZO-1) and imaging of cell morphology using scanning electron microscopy (SEM). The impact of CuO NMs and CuSO on the viability of differentiated cells was performed via assessment of cell number (DAPI staining), and visualisation of cell morphology (light microscopy). Interleukin-8 (IL-8) production by undifferentiated and differentiated Caco-2 cells following exposure to CuO NMs and CuSO was determined using an ELISA. The copper concentration in the cell lysate, apical and basolateral compartments were measured with Inductive Coupled Plasma Optical Emission Spectrometry (ICP-OES) and used to calculate the apparent permeability coefficient (P ); a measure of barrier permeability to CuO NMs. For all experiments, CuSO was used as an ionic control. CuO NMs and CuSO caused a concentration dependent decrease in cell viability in undifferentiated cells. CuO NMs and CuSO translocated across the differentiated Caco-2 cell monolayer. CuO NM mediated IL-8 production was over 2-fold higher in undifferentiated cells. A reduction in cell viability in differentiated cells was not responsible for the lower level of cytokine production observed. Both CuO NMs and CuSO decreased TEER values to a similar extent, and caused tight junction dysfunction (ZO-1 staining), suggesting that barrier integrity was disrupted. CuO NMs and CuSO stimulated IL-8 production by Caco-2 cells, decreased barrier integrity and thereby increased the P and translocation of Cu. There was no significant enhancement in potency of the CuO NMs compared to CuSO . Differentiated Caco-2 cells were identified as a powerful model to assess the impacts of ingested NMs on the GI tract.