Abstract: NtCycB2 is involved in the regulation of occurrence and development of tobacco glandular trichomes, and can response to a variety of abiotic stresses. In order to investigate the effect of NtCycB2 expression on the drought tolerance of tobacco plants, ko (NtCycB2 knockout, a high trichome density line), oe (NtCycB2 overexpression, a low trichome density line) and the tobacco cultivar K326 were used as experimental materials to compare their drought resistances under different concentrations of PEG-6000 in test tubes and drought stress in floating seedling trays. The results showed that compared with K326, the growing situation of ko line under different concentrations of PEG-6000 stress was significantly better and the stomatal aperture was significantly lower than those of K326. The biomass and chlorophyll contents of ko line were higher than those of K326, while the contents of dead cells and reactive oxygen species were lower under drought stress. The activities of antioxidant enzymes (SOD, POD) and the content of proline (Pro) of ko line were higher, while the content of MDA significantly decreased and the dehydration rate of detached leaves was significantly lower than that of K326. RT-PCR results indicated that the expression levels of NtPR1a, NtDREB2C and NtNCED5 were all significantly higher than those of K326. However, oe wilted seriously and had a higher stomatal aperture than that of K326, and the contents of dead cells and reactive oxygen species were higher. While the biomass, chlorophyll content, SOD, POD activity and Pro content were significantly lower than those of K326. MDA content and dehydration rate of detached leaves were higher than K326. The expression levels of NtPR1a, NtDREB2C and NtNCED5 were significantly lower than those of K326. Therefore, the knockout of NtCycB2 could enhance drought resistance through a series of physiological and biochemical reactions and the increase of the expression of drought related genes, while NtCycB2 overexpression could reduce the drought resistance, which indicated that the expression level of NtCycB2 played an important role in regulating tobacco drought resistance.