By covering tobacco plants with white, red, yellow, blue and purple films to obtain different light quality, the effects of light quality on anatomical structure, carboxylase activity of ribulose 1,5-biphosphate carboxylase oxygenase (Rubisco), and expression of rbc and rca genes were investigated in tobacco leaves aged from 7 to 70 days. Compared to the leaves grown under yellow film, red, blue and purple film treatments increased the leaf thickness, palisade and spongy parenchyma thickness, and density of palisade cells, but lowered the tissue interspace in the tobacco leaves. In addition, the leaves with red, blue and purple film treatments showed higher carboxylase activity of Rubisco and net photosynthetic rate and stronger expression of rbc and rca genes than those with yellow film treatment. These results indicated that, different light quality obviously influenced the anatomical structure, carboxylase activity of Rubisco and further photosynthesis efficiency of tobacco leaves, and there was a possible relationship among light quality, anatomical structure and photosynthesis efficiency of tobacco leaves to some extent.