Nitrogen availability and uptake levels can affect nutrient accumulation in plants. In this study, the effects of valine and urea supplementation on the growth of new shoots, lignin content, and carbon and the nitrogen metabolism of 'Ruiguang 39/peach' were investigated. Relative to fertilization with urea, the application of valine inhibited shoot longitudinal growth, reduced the number of secondary shoots in autumn, and increased the degree of shoot lignification. The application of valine also increased the protein level of sucrose synthase (SS) and sucrose phosphate synthase (SPS) in plant leaves, phloem, and xylem, thereby increasing the soluble sugar and starch content. It also resulted in an increase in nitrate reductase (NR), glutamine synthase (GS), and glutamate synthase (GOGAT) protein levels, with an increase in plant contents of ammonium nitrogen, nitrate nitrogen, and soluble proteins. Although urea application increased the protein level of carbon- and nitrogen-metabolizing enzymes, the increase in plant growth reduced the overall nutrient accumulation and lignin content per unit tree mass. In conclusion, the application of valine has a positive effect on increasing the accumulation of carbon and nitrogen nutrients in peach trees and increasing the lignin content.