Smoking is one of the major contributors to airway injuries. N-acetylcysteine (NAC) has been proposed as a treatment or preventive measure for such injuries. However, the exact nature of the smoking-induced injury and the protective mechanism of NAC are not yet fully understood. Here, patient tissue-derived airway organoids for modeling smoking-induced injury, therapeutic investigation, and mechanism studies are developed. Airway organoids consist mainly of ciliated cells, together with basal cells, goblet cells, and myofibroblast-like cells. The organoids display apical-out and basal-in polarity and are enriched in beating cilia, which are sensitive to smoking challenge and NAC treatment. An algorithm is developed to measure ciliary beating activity by analyzing the altered beating pattern of cilia in response to nicotine challenge and NAC treatment. Nicotinic acetylcholine receptors (nAChRs) expressed by airway organoids are involved in the mechanisms of nicotine-induced injury through the nicotine-nAChR pathway. In contrast to the common understanding that NAC has an antioxidative effect that mitigates airway damage, it is elucidated that NAC binding to nicotine can abolish the binding capacity of nicotine to nAChRs and thus prevent nicotine-induced injury. This study focuses on the advances and potential of humanized organoids in understanding biological processes, mechanisms, and identifying therapeutic targets.