Nitrogen deficiency is a major factor that affects the yield and quality of horticultural crops. Differentially expressed proteins were iden-tified in cucumber fruit under nitrogen deficiency treatment, and are expected to be highly useful for dissecting carbon, nitrogen and ascor-bate metabolism in cucumber and for improving nitrogen fertilizer utilization. Cucumber plants grown hydroponically in Hoagland solution were transferred to nitrogen-free conditions for 3 or 5 d after the blossom of female flowers. Fruit proteome profiles were assessed using two-dimensional electrophoresis. The relative expression of the genes encoding 50 selected differentially expressed proteins and 4 key enzymes in carbon metabolism was analyzed by qRT-PCR. Resulted showed that most of the identified differentially expressed proteins were mainly as-sociated with carbon metabolism, amino acid biosynthesis, ascorbate metabolism, and proteasomes. Glucose phosphorylation was enhanced, while the pentose phosphate pathway was inhibited. Carbon metabolism and the synthesis of most amino acids was affected significantly, and ascorbate might be accumulated under nitrogen deficiency in cucumber fruit.