Abstract: To explore the alleviation mechanism of exogenous spermidine (Spd) on the growth of plant under osmotic stress, hydroponic experiments were conducted on flue-cured tobacco of cv. Yuyan 6 in laboratories to study the effects of exogenous Spd at different concentrations (0, 0.2, 0.4 and 0.6 mmol·L^-1). Plant growth, active oxygen metabolism, antioxidant enzymes, relative chlorophyll content(Soil and Plant Analyzer Development, SPAD) and photosynthetic fluorescence characteristics of flue-cured tobacco seedlings under osmotic stress were simulated by polyethylene glycol (PEG-6000). The results showed that osmotic stress inhibited the growth of tobacco seedlings, while spraying exogenous Spd on leaves significantly increased the biomass, root length, root surface area, root average diameter and root tip number of tobacco seedlings. In addition, the treatment enhanced the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), reduced O₂^·- producing rate and malondialdehyde (MDA) content, and raised the relative chlorophyll content, net photosynthetic rate (P_n), transpiration rate (T_r), stomatal conductance (G_s), maximal photochemical efficiency of PSⅡ (F_v/F_m), actual photochemical efficiency of PSⅡ (φ_PSⅡ) and photochemical quenching coefficient (qP), and decreased intercellular carbon dioxide concentration (C_i) and non-photochemical quenching coefficient (NPQ) of tobacco seedlings under osmotic stress; especially Spd at the concentration of 0.4 mmol·L^-1. Therefore, exogenous Spd could effectively improve the antioxidant capacities of flue-cured tobacco seedlings under osmotic stress, reduce the accumulation of reactive oxygen radicals to maintain the stability of cell membrane, promote root development, photochemical electron transport efficiency and photosynthesis; thereby the damage of flue-cured tobacco seedlings caused by osmotic stress alleviates.