Cyclocarya paliurus has traditionally been used in medicines and nutraceutical foods. The aims of this study were to determine whether flavonoid accumulation in C. paliurus is dependent on nitrogen (N) availability and to investigate the internal C (carbon)/N balance under controlled conditions. One-year-old seedlings were grown under five increasing available N level treatments (N1-5) and were harvested throughout the 15-d experimental period. The greatest total chlorophyll amount and photosynthetic rate were achieved during the intermediate N treatments (N3 and N4). The greatest starch level was detected in N3. The total C level was relatively stable, but the total N and free amino acid levels increased, which resulted in a decreased C:N ratio. The flavonoid contents in roots and stalks decreased, while leaves showed a different pattern (peaking in N3). The flavonoid level was closely correlated with flavanone-3-hydroxylase activity, which displayed a similar variation pattern, and their levels were significantly positively correlated with those of total C and starch. Thus, the partitioning of C among primary and secondary metabolisms could be responsible for flavonoid biosynthesis and provide the basis for maintaining high yields, which increases the nutritional values of crops and medicinal plants.