Key biological functions involved in cell survival have been studied to understand the difference between the impact of exposure to TiO₂ nanoparticles (TiO₂-NPs) and their bulk counterparts (bulk-TiO₂). By selecting a unicellular eukaryotic model organism and applying proteomic analysis an overview of the possible impact of exposure could be obtained. In this study, we investigated the early response of unicellular eukaryotic protozoan Tetrahymena thermophila exposed to TiO₂-NPs or bulk-TiO₂ particles at subtoxic concentrations for this organism. The proteomic analysis based on 2DE + nLC-ESI-MS/MS revealed 930 distinct protein spots, among which 77 were differentially expressed and 18 were unambiguously identified. We identified alterations in metabolic pathways, including lipid and fatty acid metabolism, purine metabolism and energetic metabolism, as well as salt stress and protein degradation. This proteomic study is consistent with our previous findings, where the early response of T. thermophila to subtoxic concentrations of TiO₂ particles included alterations in lipid and fatty acid metabolism and ion regulation. The response to the lowest TiO₂-NPs concentration differed significantly from the response to higher TiO₂-NPs concentration and both bulk-TiO₂ concentrations. Alterations on the physiological landscape were significant after exposure to both nano- and bulk-TiO₂; however, no toxic effects were evidenced even at very high exposure concentrations. This study confirms the relevance of the alteration of the lipid profile and lipid metabolism in understanding the early impact of TiO₂-NPs in eukaryotic cells, for example, phagocytosing cells like macrophages and ciliated cells in the respiratory epithelium.