P25 Degussa represents a type of titanium dioxide (TiO2) used as a white pigment in various applications, including coatings, plastics, paper, inks and cosmetics. It is generally considered to be non-toxic, but there have been concerns about the potential skin and inhalation toxicity of TiO2 nanoparticles, as they can cause oxidative stress and other cellular damage in high quantities. A 3D skin barrier system represented by human epidermis reconstructed from highly differentiated keratinocytes cultured at the air-liquid interface (EpiDerm^TM) was used in our study to test the cytotoxicity of P25 Degussa particles. These were characterized from the point of view of structure and morphology, confirming the physico-chemical characteristics of TiO2 particles. A 24-hour exposure of cell culture to a concentration of 10 µg/mL P25 Degussa did not significantly alter cell viability compared to the control as measured by the MTT assay. It also did not induce the release of an appreciable amount of nitric oxide or lactate dehydrogenase in the extracellular environment, the values ​​being close to those of the control. Instead, an increase in the level of the cytokine IL-8 was observed, being suggestive for the initiation of an inflammatory process. Microscopy analysis of cell morphology (through hematoxylin-eosin staining and fluorescent actin labeling) revealed slight changes, especially at the apical surface of the tissue, noting some P25 Degussa particles that remained attached to this surface and were not internalized by cells. Trans-epithelial electrical resistance (TEER) measurement showed that cell barrier integrity was not altered by exposure to TiO2 particles, the tight junctions and normal cell permeability being maintained. In conclusion, our results showed that the toxicity of P25 Degussa particles in small concentration was minimal on the in vitro 3D model of the human skin, not penetrating this biological barrier.