More attention has been recently paid to the ecotoxicity of titanium dioxide nanoparticles (nano-TiO) owing to its common use in many fields. Although previous studies have shown that nano-TiO is neurotoxic, the mechanism is still largely unknown. In the present study, zebrafish embryos were exposed to 0.01, 0.1, and 1.0 mg/L nano-TiO and 1.0 mg/L micro-TiO for up to 6 days post-fertilization (dpf). Exposure to 1.0 mg/L nano-TiO significantly decreased the body length and weight of zebrafish larvae; however, the hatching and mortality rate of zebrafish embryos did not change. Behavioral tests showed that nano-TiO exposure significantly reduced the swimming speed and clockwise rotation times of the larvae. The results revealed that nano-TiO treatment adversely affected motor neuron axon length in Tg (hb9-GFP) zebrafish and decreased central nervous system (CNS) neurogenesis in Tg (HuC-GFP) zebrafish. Additionally, real-time polymerase chain reaction analysis demonstrated that genes associated with neurogenesis (nrd and elavl3) and axonal growth (α1-tubulin, mbp, and gap43) were significantly affected by nano-TiO exposure. In conclusion, our study demonstrated that early-life stage exposure of zebrafish to nano-TiO causes adverse neural outcomes through the inhibition of neurodevelopment and motor neuron axonal growth.