Although gold nanoparticles (AuNPs) are currently used in several industrial products and biomedical applications, information about their biological effects is very limited. Thus, it is becoming crucial to assess their safety and adequately investigate the complexity of cell-nanoparticles interactions. In this work, the Balb/3T3 mouse fibroblast cell line was selected as an in vitro model to study AuNPs effects. Alteration of cellular processes and biochemical pathways caused by AuNPs exposure was investigated by analysing the differentially expressed proteome. The strength of this investigation resides in combining the high-resolving power of fluorescence two-dimensional differential gel electrophoresis with protein identification by high-resolution mass spectrometry. Of interest was the difference observed in the protein pattern expression of cells exposed to 5 and 15 nm AuNPs. From 2D gel-based proteomic data, it was found that 88 and 83 proteins were de-regulated after exposure to 5 and 15 nm AuNPs, respectively. Analysis of the proteome revealed that AuNPs triggers several pathways related to cellular growth and proliferation, cell morphology, cell cycle regulation, cellular function and maintenance, oxidative stress, inflammatory response.