Wheat leaf rust, caused by Puccinia triticina, poses a growing threat to global wheat production, necessitating alternative strategies for effective disease management. This study investigated the potential of gamma-aminobutyric acid (GABA) to enhance resistance to leaf rust in two wheat cultivars: the susceptible Morocco and moderately resistant Sakha 94 cultivar. Our findings revealed that GABA significantly improved resistance in both cultivars to P. triticina, particularly in Morocco, by mitigating disease severity and reducing pustule density and size while extending both incubation and latent periods. This study assessed the effectiveness of two GABA application methods: plants received 1 mM GABA treatment, as a foliar spray, twenty-four hours prior to infection (pre-GABA), and plants received 1 mM GABA treatment both 24 h before and after infection (pre-/post-GABA), with the latter yielding significantly better results in reducing infection severity and improving plant resilience. Additionally, GABA application influenced stomatal behavior, promoting closure that may enhance resilience against leaf rust. GABA application on plants also modulated the production of reactive oxygen species (ROS). This led to a stronger oxidative burst in both susceptible and moderately resistant cultivars. GABA increased O2●− levels in guard cells and surrounding stomata, enhancing stomatal closure and the hypersensitive response. GABA enhanced the accumulation of soluble phenols and increased the activity of key antioxidant enzymes, catalase (CAT) and peroxidase (POX), which are vital for managing oxidative stress. To the best of our knowledge, this investigation represents the first report into the impact of GABA on wheat leaf rust disease.