Plants can change leaf forms, adjusting light conditions on their adaxial and abaxial surfaces, to adapt to light environments and enhance their light use efficiencies. The difference between photosynthesis on the two leaf sides (dorsoventral asymmetry) is an important factor that affects light use efficiency. However, photosynthetic dorsoventral asymmetry is rarely compared under direct and diffuse light conditions. To estimate the impacts of recently reported alterations in direct and diffuse light in the sky radiation on plant carbon assimilation, variations in morphology between the two leaf sides in tobacco (Nicotiana tabacum L.) were investigated, and the dorsoventral responses of photosynthesis to illuminating directions were compared in direct and diffuse light. Dorsoventral asymmetry was reflected in stomatal densities, anatomic structures, and photochemical traits, which caused markedly different photosynthetic rates as well as stomatal conductances both in direct and diffuse light. However, the degree of photosynthetic asymmetry was weakened in diffuse light. The diffuse light caused a greater stomatal conductance on the abaxial side than direct light, which resulted in reduced photosynthetic asymmetry. In addition, the photosynthetic dorsoventral asymmetry could be affected by the photosynthetic photon flux density. These results contribute to understanding the dorsoventral regulation of photosynthesis in bifacial leaves, and provide a reference for breeding to cope with the increase in the proportion of diffuse light in the future.