The widespread use of titanium dioxide nanoparticles (TiO₂ NPs) is gradually increasing the risk of exposure to these potentially hazardous materials. Although numerous health effects of TiO₂ NPs have been investigated, it remains unknown whether they could affect the respiratory cellular epigenome. We explored the viability, membrane integrity, intracellular ROS and genomic DNA methylation of human respiratory cells, as well as their expression of methylation-related genes, after treatment with TiO₂ NPs with diameters of 25 nm (nanotube morphology) or 60 nm (anatase morphology). Two cell lines relevant to inhalation exposure, namely human bronchial epithelial cell line (16HBE) and human non-small cell lung cancer cells (A549), were tested, with treatment concentrations ranging from 0.1 to 100 μg mL⁻¹. The TiO₂ NPs induced time- and concentration-dependent decreases in cell viability in both A549 and 16HBE cells. The reduction in cell viability was greater for the smaller particles (size 25 nm) of the nanotube type. Cellular membrane integrity assays revealed that 16HBE cells were less sensitive to TiO₂ NPs-25 nm (nanotube-type) than were A549 cells, as higher concentrations were required for cytotoxicity against the former. TiO₂ NPs-25 nm (nanotube-type) showed greater toxicity against both cell lines than TiO₂ NPs-60 nm (anatase-type). Intracellular ROS levels in both A549 and 16HBE cell were increased by TiO₂ NPs whereas pretreatment with the antioxidant N-acetyl-L-cysteine eliminated TiO₂ NPs-induced ROS accumulation and reduced cell death. Moreover, the anatase-type TiO₂ NPs resulted in decreased global DNA methylation and altered expression levels of methylation-related genes and proteins, suggesting that these NPs induce cellular epigenomic toxicity. These results allowed us to confirm the epigenetic mechanism by which TiO₂ NPs damage human respiratory cells.