Mycoplasma gallisepticum causes chronic respiratory disease in chickens, but the molecular pathways underlying the chronic pathological changes caused by M. gallisepticum in the tracheal mucosa are poorly understood, and the types of immune responses induced after infection or vaccination are not well differentiated. M. gallisepticum ts-304 is a novel live vaccine strain that is safe and more efficacious than the current commercial vaccine strain ts-11. The transcriptional profiles of the tracheal mucosa were analysed in chickens vaccinated with ts-304 at 4 and 57 weeks after vaccination and compared with those of unvaccinated chickens after experimental infection with M. gallisepticum. Pathological changes and immune cells accumulating in the mucosa were assessed by histopathological examination and indirect immunofluorescence staining. The levels of transcription of a panel of chicken cytokine and chemokine genes were quantified using RT-qPCR. Two weeks after challenge, genes, pathways, gene ontologies and protein classes involved in inflammation, cytokine production, and signaling and cell proliferation were upregulated, while those involved in formation and movement of cilia and formation of the cytoskeleton and intercellular junctional complexes were downregulated in the challenged-only birds compared to vaccinated-only, vaccinated-and-challenged, and negative-control birds. There was no significant difference in gene expression between vaccinated birds and the negative control group. After infection, there was a significant increase in the tracheal mucosal thickness and transcription of genes for cytokines and chemokines, including those for IFN-γ, IL-17, RANTES and CXCL-14, and B-cells, T-cells and macrophages accumulated in the mucosa of challenged-only birds. There were indications that acquired immunity was developing towards the end of the second week after infection - predominantly a Thelper-1 cell-dependent B cell response. The new ts-304 vaccine protects the birds against the adverse effects caused by M. gallisepticum in the tracheal mucosa for at least 57 weeks after vaccination.