Aluminium (Al) toxicity in acidic soils limits cereal productivity, yet the stomatal activities linking Al exposure to reduced growth performance remain poorly defined. Here, we investigated stomatal responses to long-term (acidic soil condition, pH 4.3) and transient Al exposure (250 µmol L⁻¹ Al³⁺, hydroponic, pH 4.3) using two barley near isogenic lines (NILs): RGT Planet (acid soil-sensitive) and P33-1 (acid soil-tolerant, introgressed with Al exclusion gene, HvAACT1). In RGT Planet, excessive Al accumulation in shoots reduced stomatal size but increased stomatal sensitivity and closure to exogenous abscisic acid (ABA). Physiological observations revealed rapid stomatal closure in RGT Planet under transient 250 µmol L⁻¹ Al exposure. Conversely, P33-1 exhibited less Al accumulation in leaves, epidermis and stomata, along with enhanced stomatal opening during light transitions, thus facilitating optimal photosynthetic performance. Molecular investigations indicated that Al induced stomatal closure involves ABA signaling pathways, reactive oxygen species (ROS), cytosolic Ca²⁺ signals, and ion channel activities in guard cells. We propose that Al susceptibility in RGT Planet is associated with excessive Al accumulation in shoots which caused reductions in stomatal size and conductance, ultimately compromising photosynthetic efficiency despite rapid stomatal responsiveness.