The Haemophilus parasuis respiratory tract pathogen poses a severe threat to the swine industry despite available antimicrobial therapies. To gain a more detailed understanding of the molecular mechanisms underlying H. parasuis response to tilmicosin treatment, microarray technology was applied to analyze the variation in gene expression of isolated H. parasuis SH0165 treated in vitro with subinhibitory (0.25 μg/ml) and inhibitory (8 μg/ml) concentrations. Tilmicosin treatment induced differential expression of 405 genes, the encoded products of which are mainly involved in the heat shock response, protein synthesis, and intracellular transportation. The subinhibitory and inhibitory concentrations of tilmicosin induced distinctive gene expression profiles of shared and unique changes, respectively. These changes included 302 genes mainly involved in protein export and the phosphotransferase system to sustain cell growth, and 198 genes mainly related to RNA polymerase, recombination, and repair to inhibit cell growth. In silico analysis of functions related to the differentially expressed genes suggested that adaptation of H. parasuis SH0165 to tilmicosin involves modulation of protein synthesis and membrane transport. Collectively, the genes comprising each transcriptional profile of H. parasuis response to tilmicosin provide novel insights into the physiological functions of this economically significant bacterium and may represent targets of future molecular therapeutic strategies.