“Plant factory with artificial lighting” (PFAL) refers to a plant production facility that can achieve mass production of vegetables year round in a controlled environment. However, the high-density planting pattern in PFALs causes low light conditions in the lower canopy, leading to leaf senescence in the outer leaves and thus to reductions in plant yields. In the present study, the effect of supplemental upward lighting underneath the plants on photosynthetic characteristics and plant yield was examined in lettuce, in comparison with supplemental downward lighting from above the plants at the same light intensity. Supplemental upward lighting increased the curvature factor of the photosynthetic response to light from above the plants. Moreover, supplemental upward lighting significantly enhanced the lettuce yield by retarding the senescence of the outer leaves. Here, we propose a novel cultivation system with a combination of downward lighting and supplemental upward lighting that can effectively increase plant growth and yield in PFALs.