Previous research showed that nano-TiO2 could significantly promote photosynthesis and greatly improve growth of spinach, but we also speculated that an increase of spinach growth by nano-TiO2 treatment might be closely related to the change of nitrogen metabolism. The effects of nanoanatase TiO2 on the nitrogen metabolism of growing spinach were studied by treating them with nano-anatase TiO2. The results showed that nano-anatase TiO2 treatment could obviously increase the activities of nitrate reductase, glutamate dehydrogenase, glutamine synthase, and glutamic-pyruvic transaminase during the growing stage. Nano-anatase TiO2 treatment could also promote spinach to absorb nitrate, accelerate inorganic nitrogen (such as NO3--N and NH4+-N) to be translated into organic nitrogen (such as protein and chlorophyll), and enhance the fresh weight and dry weights.