Soil moisture was closely linked to plant photosynthesis rate and plant productivity. Water stress was important factors for photosynthetic depression and yield decrease. However, the key limiting step and underlying mechanism was highly uncertain. The resistance distribution along the pathway of CO2 transport from the atmosphere surrounding the leaf to the site of carboxylation inside the chloroplast stroma of tomato under different soil water content gradients was explored. Soil moisture was maintained by a standardized gravimetric approach. Stomatal and mesophyll conductance were estimated from simultaneous measurement of leaf gas exchange and chlorophyll fluorescence. The results showed that the photosynthesis rate (Pn), rubisco carboxylation capacity (Vc,max), maximum electron transport capacity (Jmax) and carboxylation efficiency (CE) were increased with the increase of soil moisture, which showed as“S” curves and can be described in logistic models. Stomatal conductance, mesophyll conductance and the total conductance for CO2 transport were decreased with water stress. The proportions of stomatal and mesophyll conductance limitations imposed on photosynthetic depression were increased with soil water stress, which were the dominant limiting factors;in contrast to stomatal and mesophyll limitation, biochemical limitations were increased with the increase of soil moisture and performed as predominant limiting factors when soil moisture was sufficient. Stomatal conductance showed positive linear relationship with leaf water potential, which was declined with soil water stress;mesophyll conductance showed negative linear relationship with leaf mass area, which was increased with soil water stress. The research result demonstrated that stomatal and mesophyll resistance for CO2 uptake were key limiting step for photosynthesis rate. The greatest resistance of stomata under water stress was determined by the turgor loss of guard cells while the mesophyll conductance was determined by the leaf anatomical structure.