Medicago truncatula is one of the most studied model plants. Nevertheless, the genome of this legume remains incompletely determined. We used RNA-Seq to characterize the transcriptome during the early organogenesis of the nodule and during its functioning. We detected 37,333 expressed transcription units (TUs), 1,670 had never been described before and were functionally annotated. We identified 7,595 new transcribed regions, mostly corresponding to 5’ and 3’ UTR extensions and new exons associated with 5,264 previously annotated genes. We also inferred 23,165 putative transcript isoforms from 6,587 genes and measured the abundance of transcripts for each isoform, which suggests an important role for alternative splicing in the generation of proteome diversity in M. truncatula. Finally, we carried out a differential expression analysis, which provided a comprehensive view of transcriptional reprogramming during nodulation. In particular, depletion of nitric oxide in roots inoculated with Sinorhizobium meliloti greatly increased our understanding of the role of this reactive species in the optimal establishment of the symbiotic interaction, in revealing differential patterns of expression for 2,030 genes and, in pointing to the inhibition of the expression of defense genes.