Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs, and the causative agent of Glässer's disease. This disease is characterized by polyserositis and arthritis, produced by the severe inflammation caused by the systemic spread of the bacterium. After an initial colonization of the upper respiratory tract, H. parasuis enters the lung during the early stages of pig infection. In order to study gene expression at this location, we sequenced the ex vivo and in vivo H. parasuis Nagasaki transcriptome in the lung using a metatranscriptomic approach. Comparison of gene expression under these conditions with that found in conventional plate culture showed generally reduced expression of genes associated with anabolic and catabolic pathways, coupled with up-regulation of membrane-related genes involved in carbon acquisition, iron binding and pathogenesis. Some of the up-regulated membrane genes, including ABC transporters, virulence-associated autotransporters (vtaAs) and several hypothetical proteins, were only present in virulent H. parasuis strains, highlighting their significance as markers of disease potential. Finally, the analysis also revealed the presence of numerous antisense transcripts with possible roles in gene regulation. In summary, this data sheds some light on the scarcely studied in vivo transcriptome of H. parasuis, revealing nutritional virulence as an adaptive strategy for host survival, besides induction of classical virulence factors.