Titanium dioxide (TiO 2 ) nanoparticles (NPs) are manufactured worldwide for a variety of engineering and bioengineering applications. TiO 2 NPs are frequently used as a material for orthopedic implants. However, to the best of our knowledge, the biocompatibility of TiO 2 NPs and their effects on osteoblast cells, which are responsible for the growth and remodeling of the human skeleton, have not been thoroughly investigated. In the research reported here, we studied the effects of exposing hFOB 1.19 human osteoblast cells to TiO 2 NPs (5–15 nm) for 24 and 48 hours. Cell viability, alkaline phosphatase (ALP) activity, cellular uptake of NPs, cell morphology, superoxide anion (O 2 •−2 ) generation, superoxide dismutase (SOD) activity and protein level, sirtuin 3 (SIR3) protein level, correlation between manganese (Mn) SOD and SIR, total antioxidant capacity, and malondialdehyde were measured following exposure of hFOB 1.19 cells to TiO 2 NPs. Exposure of hFOB 1.19 cells to TiO 2 NPs resulted in: (1) cellular uptake of NPs; (2) increased cytotoxicity and cell death in a time- and concentration-dependent manner; (3) ultrastructure changes; (4) decreased SOD and ALP activity; (5) decreased protein levels of SOD1, SOD2, and SIR3; (6) decreased total antioxidant capacity; (7) increased O 2 •− generation; and (8) enhanced lipid peroxidation (malondialdehyde level). The linear relationship between the protein level of MnSOD and SIR3 and between O 2 •− content and SIR3 protein level was observed. Importantly, the cytotoxic effects of TiO 2 NPs were attenuated by the pretreatment of hFOB 1.19 cells with SOD, indicating the significant role of O 2 •− in the cell damage and death observed. Thus, decreased expression of SOD leading to increased oxidizing stress may underlie the nanotoxic effects of TiO 2 NPs on human osteoblasts.