Objectives of this study were to clarify the involvement of root to shoot ABA in regulation of leaf stomatal conductance (gs) and mesophyll conductance (gm) during progressive soil drought, and to investigate its impact on leaf photosynthesis and intrinsic water use efficiency (WUEi) in pot-planted tomato. A fast-stomatal closure was related to decrease of Ψleaf in the early stage of soil drought, whereas gm kept unchanged until ABAxylem synchronously increased at threshold of relative soil water content (RSWC) decreasing from 51.43 to 44.69%. This out-of-step response between gs and gm resulted in an increase of gm/gs ratio and potentially improved WUEi during RSWC decreased from 51.43 to 40.16%. Meanwhile, gs and gm declined logarithmically with increasing xylem sap ABA concentration (p<0.01). The sensitivity of gm response to root to shoot ABA signaling, even though less than gs, played a key role in regulation of CO2 diffusion into the chloroplast as soil drying proceeded. gs mainly limited leaf photosynthesis with RSWC in range of 51.43 to 40.16%, and relative contribution of gm limitation exceed that of gs limitation as soil further dried. These results provided new understanding of ABA in the regulation of gm and WUEi in response to drought stress.