Being a proven photocatalyst, nano-anatase is capable of undergoing electron transfer reactions under light. In previous studies, we had proven that nano-anatase improved photosynthesis and greatly promoted spinach growth. However, the molecular mechanisms of carbon reaction promoted by nano-anatase are still not clearly understood. In the present paper, reverse transcription polymerase chain reaction and Northern blotting were carried out, and the results showed that the expression of ribulose-1, 5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (rbcS) and Rubisco large subunit (rbcL) messenger RNA (mRNA) in the nano-anatase-treated spinach were obviously promoted, respectively, while the effects of bulk TiO2 were not so significant. Accordingly, the protein expression of Rubisco from the nano-anatase-treated spinach was increased by 40% compared with control. Further analysis indicated that the activity of Rubisco in the nano-anatase-treated spinach was significantly higher than the control, by up to 2.33 times, and bulk TiO2 treatment had no such significant effects. Together, one of the molecular mechanisms of carbon reaction promoted by nano-anatase is that the nano-anatase treatment results in the enhancement of Rubisco mRNA amounts, the protein levels, and activity of Rubisco, thereby leading to the improvement of Rubisco carboxylation and the high rate of photosynthetic carbon reaction.