Titanium dioxide nanoparticles are increasingly being used in pharmaceutical and cosmetic products. The high aspect ratio of fibrous nanomaterials, such as carbon nanotubes and TiO2 nanofibers (TiO2NFs), similar to the one used in this study makes them an attractive structural material and has attracted a lot of attention due to their possible negative health effects as suggested by their morphological similarities with asbestos. In the present study, therefore, toxicity of TiO2NFs was evaluated in human cervical adenocarcinoma HeLa cells. The TEM and XRD analyses showed that TiO2NFs used in this study are pure with uniform diameter of around 200 nm, and their length to width aspect ratio ranged between 5 and 15. Exposure of HeLa cells to TiO2NFs induced significant cytotoxicity even at doses as low as 2 μg/ml. The intracellular uptake of TiO2NFs in cells was shown by Alizarin Red S (ARS) labeled nanofibers. The mechanism of toxicity is mainly due to the induction of cellular oxidative stress, as revealed by elevated ROS levels, reduced antioxidant levels, and increased lipid peroxidation leading to apoptosis. The cell cycle analysis indicated G2/M cell cycle arrest in the cells exposed to TiO2NF. TiO2NFs treatment to HeLa cells resulted in increased expression of proapoptotic proteins Bax with an increase in cytosolic Cytochrome-C and inhibition of anti-apoptotic protein Bcl-2. Our results revealed the potential mechanism of cellular effects of TiO2NFs.