Titanium dioxide (TiO₂) is manufactured worldwide as crystalline and amorphous forms for multiple applications, including tissue engineering, but our study proposes analyzing the impact of crystalline phases of TiO₂ on Mesenchymal Stem Cells (MSCs). Several studies have already described the regenerative potential of MSCs and TiO₂ has been used for bone regeneration. In this study, polydispersity index and sizes of TiO₂ nanocrystals (NCs) were determined. Adipose tissue-derived Mesenchymal Stem Cells (AT-MSCs) were isolated and characterized in order to evaluate cellular viability and the internalization of nanocrystals (NCs). All of the assays were performed using the TiO₂ NCs with 100% anatase (A), 91.6% anatase/9.4% rutile (AR), 64.6% rutile/35.4% anatase (RA), and 84.0% rutile/16% brookite (RB), submitted to several concentrations in 24-h treatments. Cellular localization of TiO₂ NCs in the AT-MSCs was resolved by europium-doped NCs. Viability was significantly improved under the predominance of the rutile phase in NCs with localization restricted at the cytoplasm, suggesting that AR and RA NCs are not genotoxic and can be associated with most cellular activities and metabolic pathways, including glycolysis and cell division.