Omics technologies, such as proteomics or metabolomics, have to date been applied in the field of nanomaterial safety assessment to a limited extent. To address this dearth, we developed an integrated approach combining the two techniques to study the effects of two sizes, 5 and 30 nm, of gold nanoparticles (AuNPs) in Caco-2 cells. We observed differences in cells exposed for 72 h to each size of AuNPs: 61 responsive (up/down-regulated) proteins were identified and 35 metabolites in the cell extract tentatively annotated. Several altered biological pathways were highlighted by integrating the obtained multi-omics data with bioinformatic tools. This provided a unique set of molecular information on the effects of nanomaterials at cellular level. This information was supported by complementary data obtained by immunochemistry, microscopic analysis and multiplexed assays. A part from increasing our knowledge on how the cellular processes and pathways are affected by nanomaterials (NMs), these findings could be used to identify specific biomarkers of toxicity or to support the safe-by-design concept in the development of new nanomedicines.