Fusarium wilt is one of the major biotic factors limiting cucumber (Cucumis sativus L.) growth and yield. The outcomes of cucumber-Fusarium interactions can be influenced by the form of nitrogen nutrition (nitrate [NO3- ] or ammonium [NH4+ ]); however, the physiological mechanisms of N-regulated cucumber disease resistance are still largely unclear. Here, we investigated the relationship between nitrogen forms and cucumber resistance to Fusarium infection. Our results showed that on Fusarium infection, NO3- feeding decreased the levels of the fungal toxin, fusaric acid, leaf membrane oxidative, organelle damage and disease-associated loss in photosynthesis. Metabolomic analysis and gas-exchange measurements linked NO3- mediated plant defence with enhanced leaf photorespiration rates. Cucumber plants sprayed with the photorespiration inhibitor isoniazid were more susceptible to Fusarium and there was a negative correlation between photorespiration rate and leaf membrane injury. However, there were positive correlations between photorespiration rate, NO3- assimilation and the tricarboxylic acid (TCA) cycle. This provides a potential electron sink or the peroxisomal H2 O2 catalysed by glycolate oxidase. We suggest that the NO3- nutrition enhanced cucumber resistance against Fusarium infection was associated with photorespiration. Our findings provide a novel insight into a mechanism involving the interaction of photorespiration with nitrogen forms to drive wider defence.