Modulation of stomatal morphology and behavior is associated with changes in water use of the plant due to varied input of water, yet the underlying mechanism of such association remains largely elusive. We investigated the effects of reduced irrigation on leaf gas exchange and stomatal morphology of tobacco (Nicotiana tabacum L.) plants grown in a Ferralsol and an Anthrosol amended by softwood (SWB) and wheat (Triticum aestivum L.) straw biochar (WSB). Compared to plants grown under full irrigation (FI), deficit irrigation (DI) and especially alternate partial root-zone drying irrigation (PRD) significantly decreased the stomatal conductance (g_s) while marginally reducing the leaf photosynthetic rate (A_n), thereby enhancing intrinsic water-use efficiency (WUE_i=A_n/g_s), whereas the effects of biochars were not evident. Moreover, reduced irrigation, particularly PRD, increased the concentration of abscisic acid in the leaf ([ABA]_leaf) compared to FI, which was further amplified by WSB addition. Leaf carbon isotope discrimination (Δ¹³C_leaf) responded similarly to irrigation and biochar addition as g_s. Compared to FI, reduced irrigation (DI and PRD) lowered stomata size (SS) but increased their density (SD) and this effect was more pronounced for PRD coupled with WSB. Moreover, SD was significantly positively correlated with [ABA]_leaf, while negatively correlated with g_s, soil water content, and Δ¹³C_leaf. The opposite correlations between SS and these variables were observed. The results collectively suggest that ABA-mediated the effects of PRD combined with WSB amendment resulted in higher SD and lower SS and g_s, contributing to the increased WUE_i and long-term WUE of tobacco plants.