Tomato seedlings were treated with nutrient solutions containing varying nitrogen concentrations (50, 150, and 250 mg·L⁻¹) after germination until the completion of flower bud differentiation. The changes in nutrient content, enzyme activity, endogenous hormone levels, and gene expression in the stem apex were analyzed to explore the mechanisms regulating the number of locules in tomatoes at different nitrogen concentrations. The results indicated that an increase in nitrogen concentration facilitated the differentiation of tomato flower buds, increased the number of fruit locules, and increased the contents of soluble sugar, soluble protein, starch, and sucrose, as well as the activities of the enzymes POD, NR, and PPO in the seedling stem apex. The contents of soluble sugars and soluble proteins, as well as the activities of POD, NR, and PPO, were closely correlated with the number of fruit locules. An increase in nitrogen concentration was also found to elevate cytokinin levels while reducing auxin content in the stem apex. The transcriptome analysis screened for peroxidase genes, auxin response genes, and cytokinin synthesis genes. The analysis of gene expression patterns suggests that CKX and LOG6 play significant roles in flower development. Additionally, combined physiological changes indicated that an increase in nitrogen concentration during the tomato seedling stage leads to a higher number of fruit locules, which may be associated with elevated cytokinin content, primarily involving the key genes CKX and LOG6.