Camptotheac acuminata Decne is a unique tree species in China with an important secondary metabolite, camptothecin (CPT), used in the treatment of cancer. Nitrogen (N) is an important element that affects plant growth and the accumulation of CPT. Reports on the effect of N on CPT synthesis from a genetic perspective are scarce. To explore the effects of different N sources and levels on CPT synthesis in C. acuminata, two-year-old seedlings were fertilized with different concentrations of pure ammonium sulphate, source of ammonium N (NH₄⁺–N), and potassium nitrate for nitrate N (NO₃⁻–N). Concentrations of 2.5, 5, 7.5, and 10 g pot⁻¹ NH₄⁺–N and NO₃⁻–N were used. The results showed that 7.5 g NH₄⁺–N and NO₃⁻–N treatments were best for growth and fresh weight of leaves. Compared with the other treatments, the CPT content, tryptophan synthase and tryptophan decarboxylase activities, and expression of the CaTSB and CaTDC1 genes under the 2.5 g NH₄⁺–N and NO₃⁻–N treatments peaked significantly at 30 days. However, the expression of CaTDC2 surpassed that of the other two genes at 60 days. Therefore, compared with NH₄⁺–N source, the NO₃⁻–N source was more beneficial for growth, and NO₃⁻–N was better for CPT yield. Consequently, leaves of C. acuminata treated with 2.5 g NO₃⁻–N could be harvested after 30 days to obtain maximum CPT content. CaTDC1 is more closely linked to CPT synthesis. The results of this study improved the production of CPT in C. acuminata via fertilization.