Abstract: To investigate the effect of long-stalked glandular trichomes on drought tolerance in tobacco, the growth performance under drought stress was compared among the flue-cured tobacco cultivar K326 and its mutants with high-density long-stalked glandular trichomes (LK326) and high-density protective trichomes (NK326). Leaf glandular trichome development, leaf surface exudate content, antioxidant enzyme activities superoxide dismutase (SOD) and peroxidase (POD), and the contents of the membrane lipid peroxidation product malondialdehyde (MDA) and the osmotic regulatory substance proline (Pro) were determined in the three materials. The expression levels of drought-tolerance-related genes in leaves were determined by RT-qPCR. The results showed that, under drought stress, LK326 exhibited the best drought-tolerance performance, as indicated by the highest biomass, the lowest leaf water loss rate, the smallest stomatal aperture, the highest chlorophyll and Pro contents as well as SOD and POD activities, the lowest MDA content, and the lowest degree of oxidative damage; K326 ranked second, and NK326 performed the worst. Before stress, LK326 had the highest density of long-stalked glandular trichomes and the largest glandular head diameter, followed by K326, whereas NK326 had almost no long-stalked glandular trichomes. After stress, the proportion of long-stalked glandular trichomes in K326 and NK326 increased significantly; meanwhile, the total leaf surface exudate contents of K326, NK326, and LK326 all increased significantly, reaching 1.55, 7.49, and 1.30 times those before drought stress, respectively. In addition, the expression levels of antioxidant-related genes (NtSOD and NtPOD) and drought-tolerance-related genes (NtNCED5 and NtDREB2C) were significantly upregulated in the three lines under drought stress; overall, the relative expression levels were higher in LK326 and lower in NK326.