The functional complexity of the Tuber melanosporum transcriptome has not yet been fully elucidated. Here, we applied high-throughput Illumina RNA-sequencing (RNA-Seq) to the transcriptome of T. melanosporum at different major developmental stages, that is free-living mycelium, fruiting body and ectomycorrhiza. Sequencing of cDNA libraries generated a total of c. 24 million sequence reads representing >882 Mb of sequence data. To construct a coverage signal profile across the genome, all reads were then aligned to the reference genome assembly of T. melanosporum Mel28. We were able to identify a substantial number of novel transcripts, antisense transcripts, new exons, untranslated regions (UTRs), alternative upstream initiation codons and upstream open reading frames. This RNA-Seq analysis allowed us to improve the genome annotation. It also provided us with a genome-wide view of the transcriptional and post-transcriptional mechanisms generating an increased number of transcript isoforms during major developmental transitions in T. melanosporum.