The objective of this experiment was to investigate the reduced glutathione (GSH) participated in the effects of photosynthesis and fluorescence regulation pathway of exogenous nitric oxide (NO) in cucumber seedling leaves under low temperature (10 ℃/6 ℃) stress. The substratecultured ‘Jinyan No.4’ cucumber seedlings were used as the test materials. Fourleaf onehearted plants were set at a photoperiod of 14 h/10 h, day and night temperature of 25 ℃/20 ℃ in a light incubator, and leaf surface pretreatment with GSH synthase inhibitors (BSO, butanamine sulfoxide amine), NADPH synthetase inhibitor (6AN, 6amino nicotinamide), and nitric oxide NO donor (SNP, sodium nitroferrocyanide) after 8 hours The daynight temperature dropped to 10 ℃/6 ℃ 24 h after the first spraying, and cucumber leaves cell membrane permeability, gas exchange and chlorophyll fluorescence parameters and other changes were measured after 24 h of cold stress. The results showed that： (1) compared with the control, the SNP treatment significantly increased the relative water content, chlorophyll content, net photosynthetic rate (P_n), transpiration rate (T_r) and stomatal conductance (G_s), Maximum photochemical efficiency (F_v/F_m) of photosystem Ⅱ (Φ_PSⅡ), the quantum yield of PSⅡ regulated energy dissipation (NPQ), photochemical quenching coefficient (qP), apparent photosynthetic electron transport rate (ETR), the relative fluorescence of the nitric oxide, the OJIP phase of rapid chlorophyll fluorescence induction curve of cucumber seedlings and maintain high fluorescence yield after 24 h of low temperature stress, significantly decreased intercellular CO₂ concentration (C_i), alleviated continual enhancement of MDA content and the quantum yield of nonregulatory energy dissipation Y_NO. (2) Compared with SNP treatments, BSO+SNP and 6AN+SNP treatments significantly reduced the effect of SNP; SNP treatment of cucumber seedling leaves the maximal photochemical efficiency of primary photochemistry (φ_Po), the quantum yield of light energy absorption by PSⅡ reaction center for electron transfer (φ_Eo) and the probability of capturing excitons for transporting electrons to other electron acceptors downstream of the electron transport chain Q_A (ψ_o), fluorescence parameters related to the acceptorside properties of the PSⅡ reaction center include the oxygen release complex (F_OK), the reduction capacity of Q_A (F_KJ), the reduction capacity of Q_B (F_IP) and the density of active reaction centers per excited crosssection (RC/CS) were significantly higher than that of CK. (3) Compared with SNP treatment, the values of φ_Po, φ_Eo, ψ_o, RC/CS, F_KJ, F_JI and F_IP were significantly decreased in BSO+SNP and 6AN+SNP treatment, while M_o and V_J increased significantly. Under low temperature stress, GSH plays an important role in exogenous NO increasing the relative water content, reducing lipid peroxidation, stabilizing photosystem Ⅱ and promoting electron transfer capability of donor side and recipient side of electron transfer chain in PSⅡ reaction center and improved the cold resistance of cucumber seedlings.