High nitrogen (N) application improves the acclimation of plant photosynthesis to water deficit stress, however, the metabolic details for this are not yet fully understood. Using hydroponics, we found that moderate water deficit simulated with 10% PEG 6000 inhibited rice photosynthetic rate by 17.2% in low N condition (LN, 0.71 mM) versus 7.9% in moderate N condition (MN, 2.86 mM). Plants grown at MN had an increased photorespiratory serine metabolism and glycerate recycling under water stress, which was in accordance with the enhancement of glutathione concentration and ascorbate-glutathione cycle, indicating that the serine metabolism plays a significant role in improving antioxidant capacity. Additionally, the up-regulation of GS2 facilitated the re-assimilation of NH₃ released in photorespiration. Aspartate aminotransferase (AspAT)- and glutamate:glyoxylate aminotransferase (GGAT)-mediated glutamate transamination and phosphoenolpyruvate carboxylase (PEPC)-mediated anaplerotic reaction provided a carbon skeleton, 2-oxoglutarate, for NH₃ assimilation, which reduced the depletion of sugars. Sugars therefore can be stored and used for the regeneration of RuBP through the pentose phosphate pathway to maintain CO₂ assimilation. In contrast, water deficit stress-induced protein degradation, down-regulation of N assimilation, and depletion of carbohydrates in LN-supplied plants, as well as the failure of the ascorbate-glutathione cycle due to reduced glutathione biosynthesis, perturbed the function of chloroplasts. We conclude that high N supply preserves the biochemistry of photosynthesis through coordinated regulation of the C and N metabolism to facilitate the acclimation of rice photosynthesis to water deficit stress.