Numerous studies have demonstrated the in vitro and in vivo neurotoxicity of nanoparticulate titanium dioxide (nano-TiO2 ), a mass-produced material for a large number of commercial and industrial applications. The mechanism of nano-TiO2 -induced inhibition of axonal development, however, is still unclear. In our study, primary cultured hippocampal neurons of 24-hour-old fetal Sprague-Dawley rats were exposed to 5, 15, or 30 μg/mL nano-TiO2 for 6, 12, and 24 hours, and the toxic effects of nano-TiO2 exposure on the axons development were detected and its molecular mechanism investigated. Nano-TiO2 accumulated in hippocampal neurons and inhibited the development of axons as nano-TiO2 concentrations increased. Increasing time in culture resulted in decreasing axon length by 32.5%, 36.6%, and 53.8% at 6 hours, by 49.4%, 53.8%, and 69.5% at 12 hours, and by 44.5%, 58.2%, and 63.6% at 24 hours, for 5, 15, and 30 μg/mL nano-TiO2 , respectively. Furthermore, nano-TiO2 downregulated expression of Netrin-1, growth-associated protein-43, and Neuropilin-1, and promoted an increase of semaphorin type 3A and Nogo-A. These studies suggest that nano-TiO2 inhibited axonal development in rat primary cultured hippocampal neurons and this phenomenon is related to changes in the expression of axon growth-related factors.