Eucalyptus species are the most widely planted hardwood trees in the world due to their high productivity, wide adaptability and valuable wood properties. Wood is a specific tissue mainly composed of lignified secondary cell walls. Lignin impairs the accessibility of cellulose during kraft pulping as well as during saccharification, a key step of bioethanol production. The application of nitrogen (N) fertilizers is one strategy to increase growth rates and productivity since nitrogen is one of the most limiting nutrients for tree growth and carbon sequestration. However, the effects of N availability on wood properties and related gene expression are poorly understood. We set up an experimental system in which rooted cuttings of Eucalyptus urophylla x grandis were fertilized during 30 days with three different amounts of N (limiting; adequate; luxuriant). For the luxuriant N treatment, we used two nutrient solutions with different concentration of NO3- and NH4+. The effects of N fertilization were studied at the phenotypic level including histochemical analysis for lignin and/or cellulose and at the whole transcriptome level using Illumina mRNA-Seq technology. We observed that genes involved in the biosynthesis of cell wall main components (lignin, cellulose and hemicelluloses) were differentially expressed between the treatments. Most of the lignin biosynthetic pathway genes were up-regulated in the treatment with less nitrogen (-N) and down-regulated in both +N treatments, confirming the observations made by histochemical stains with phloroglucinol. These results suggest that N fertilization could not only increase growth and productivity but also contribute for better wood properties.