Haemophilus parasuis is a common inhabitant of the upper respiratory tract of pigs, and the causative agent of Glässer’s disease, which is characterized by polyserositis and arthritis. After an initial colonization of the upper respiratory tract, H. parasuis has been detected in lung during the early stages of pig infection. In order to study the gene expression at this location, we tried to simulate in vivo growth by conducting a 2h incubation in lung, followed by whole transcriptome sequencing using RNA-seq technology. The lungs from a healthy pig were removed and transported to the laboratory, where they were inoculated with a suspension of the virulent strain Nagasaki. After 2h at 37ºC, bacteria were recovered by lavage and mRNA was purified and sequenced. Results were compared with conventional plate culture, revealing significant changes in some metabolic pathways, as well as expression of already reported virulence factors. Among the up-regulated genes, Gene Ontology (GO) enrichment analysis showed over-representation of membrane-related genes, some of them involved in transmembrane transport and pathogenesis, as well as biological process related to stimulus response, queuosine biosynthesis, heme transport and tryptophan biosynthesis. Some differentially expressed putative virulent factors, such as two ABC transporters, were not found in the available genomes of non-virulent strains. Among the down-regulated genes, tricarboxylic acid cycle, cysteine biosynthetisis and maltose transport were over-represented. Results were validated using RT-qPCR. Additionally, the analysis also revealed presence of numerous putative antisense transcripts (asRNA) and small RNA (sRNA). In summary, this data sheds some light on the scarcely studied transcriptome of H. parasuis and presents an ex-vivo model useful for other researchers.