Nitrogen fixation in legumes requires the development of specialized organs called root nodules. Here we characterized the high-confidence transcriptome and genome-wide patterns of H3K4me3 marks in soybean roots and mature nodules symbiotic with Sinorhizobium fredii. Changes in H3K4me3 levels were positively associated with the transcription levels of functional genes in the nodules. The up-regulation of H3K4me3 levels was not only present in leghaemoglobin and nodulin-related genes, but also in genes involved in nitrogen and carbon metabolic pathways. In addition, genes regulating the transmembrane transport of metal ions, phosphates, sulphates, peptides, and sugars were differentially modified. On the contrary, a loss of H3K4me3 marks was found in several key transcription factor genes and was correlated with the down-regulation of the defense- related network in nodules, which could contribute to nodule maintenance. All these findings demonstrate massive reprogramming of gene expressions via alterations in H3K4me3 levels in the genes in mature soybean nodules.