Abscisic acid (ABA) is important for mediating abiotic stress responses and also in plant immunity. Its involvement in the resistance of wild tobacco Nicotiana attenuata to the pathogenic fungus Alternaria alternata, tobacco pathotype, was investigated. After infection, many fungal hyphae were observed to enter leaf tissue through stomata within 24 h, and necrotic lesions were formed after 3–6 days. Importantly, source–sink transition leaves (leaf 0) were more resistant than all fully expanded ones, and this was correlated with more ABA and ABA-related gene transcripts and lower stomatal conductance in leaf 0 after infection. When supplied with ABA or incubated in the dark for 1 day, fully expanded leaves, which accumulated less ABA and showed higher stomatal conductance after infection in comparison to leaf 0, showed increased resistance. Mitogen-activated protein kinase 4-silenced plants, in which ABA-induced stomatal closure responses and resistance to A. alternata were highly impaired, did not show stomatal conductance change after infection, and leaf 0 and fully expanded leaves all developed bigger lesions than in wildtype plants. Thus the ABA signalling pathway mediated by NaMPK4 is required for the resistance of N. attenuata to A. alternata, at least in part through the stomatal closure responses.