Abstract: Using split plot and then-split plot design, effects of water-nitrogen coupling on photosynthesis and ultrastructure of cucumber (Cucumis sativus) (Jinyou No.35) under CO₂ enrichment were investigated. The main plot had two CO₂ concentrations: ambient CO₂ concentration (400 μmol·mol^-1, A) and doubled CO₂ concentration (800±20 μmol·mol^-1, E). The split plot had two treatments: no drought stress (95% of field capacity, W) and drought stress (75% of field capacity, D). The then-split plot contained low nitrogen treatment (450 kg·hm^-2, N₁) and high nitrogen treatment (900 kg·hm^-2, N₂). The results showed that under the condition of drought and high nitrogen, increasing CO₂ enhanced the cucumber plant height, and no matter what kinds of water treatment, CO₂ enrichment increased the leaf area significantly under high nitrogen. Under the condition of normal irrigation, the photosynthetic rate, stomatal conductance and transpiration rate of high nitrogen treatment were higher than low nitrogen treatment, while it was under the drought condition. Elevated CO₂ enhanced the water use efficiency of cucumber leaf which increased with increasing nitrogen application rate. Under drought stress, cucumber adaxial surface porosity density was increased, and the CO₂ enrichment and high nitrogen significantly reduced the stomatal density. Increasing nitrogen application improved the number of chloroplast, and reduced that of starch grains. Drought stress decreased the number of chloroplast, but tended to promote the number of starch grains. Drought stress increased the chloroplast length and width, and the size of the starch grains, while high nitrogen reduced the length and width of the chloroplast and starch grains. CO₂ enrichment and high nitrogen increased grana thickness and layers (except ADN₂), and the slice layer of EDN₂ was significantly higher than that of ADN₂. In conclusion, CO₂ enrichment and suitable water and nitrogen could promote the development of chloroplast thylakoid membrane system, significantly increase the thickness of grana and the number of grana lamella, and effectively improve the chloroplast structure of cucumber, which would benefit the photosynthesis of cucumber plants and ability to utilize CO₂ and water and nitrogen.