Abstract: To investigate the effect of long-stalked glandular trichomes on tobacco drought tolerance, the growth performance under drought stress was compared among the flue-cured tobacco cultivar K326, its high-density long-stalked glandular trichome mutant (LK326), and its high-density protective trichome mutant (NK326). Determinations were conducted on the three lines for leaf trichome development, trichome exudate content, antioxidant enzyme activities superoxide dismutase (SOD) and peroxidase (POD), as well as the content of the membrane lipid peroxidation product malondialdehyde (MDA) and the osmolyte proline (Pro). The expression levels of drought-tolerance-related genes in leaves were measured by qRT-PCR.The results showed that, under drought stress, LK326 exhibited the best growth performance, manifested as the highest biomass, the lowest leaf water loss rate, the smallest stomatal aperture, the highest chlorophyll content, SOD and POD activities, and Pro content, while having the lowest MDA content and the mildest oxidative damage; K326 ranked second; NK326 performed the worst. Before stress, LK326 had the highest long-stalked glandular trichome density and glandular head diameter, K326 ranked second; NK326 essentially lacked long-stalked glandular trichomes. After stress, the proportion of long-stalked glandular trichomes in both K326 and NK326 significantly increased, and their total leaf surface exudate amounts increased to 1.55-fold and 6.98-fold, respectively, compared to pre-drought levels. Furthermore, the expression levels of antioxidant-related genes (NtSOD and NtPOD) and drought-tolerance-related genes (NtNCED5 and NtDREB2C) in the three lines were significantly upregulated under drought stress, with the overall pattern being: LK326 > K326 > NK326.