Two tobacco (Nicotiana tabacum L.) cultivars (DHJ5210 and ZY100) with different drought tolerance were used to study the photosynthetic response to different water stress and the effects of glycinebetaine applied through roots on photosynthetic capacity. The physiological mechanisms of the improvement of tobacco photosynthesis by glycinebetaine applied through roots were analyzed. The results indicated that photosynthetic apparatus of tobacco leaves were more brittle and damaged by water stress, accompanied by negative changes of RWC (Fig. 2), chlorophyll content (Fig. 3), PSII photochemistry efficiency (Fig. 4), activity of Hill reaction (Fig. 5) and ATPase (Fig. 6), and the damage was more serious to ZY100 (drought sensitive cultivar) than DHJ5210 (drought resistant cultivar) under the same stress condition. Glycinebetaine applied through roots could be absorbed by tobacco roots and then accumulated in leaves (Fig. 1), which alleviated the decrease in photosynthetic variables of two cultivars and the improvement was more significant on ZY100 than DHJ5210. The positive function of glycinebetaine might be involved in its protective effect on antioxidant enzymes (Fig. 8), chloroplast protein and thylakoid membrane (Fig. 7).