Aims To determine whether cytokine production and gene expression levels vary in epithelial cells infected by virulent strains of Mycobacterium tuberculosis. Methods Supernatants were collected and RNA extracted from A549 epithelial cells infected with the KZN, Beijing, F11, F28, Unique and H37Rv strains of M. tuberculosis. Cytokines were quantified using the Bio-Plex Pro Human Cytokine 27-Plex assay (BioRad) and the Bio-Plex 200 system. Changes in gene expression were determined by whole transcriptome analysis using the Illumina HiSeq 2000 platform. The 50bp reads were mapped to the human genome (hg19) using Tophat (2.0.10). Differential expression was quantified using Cufflinks (2.1.0) and a log fold change cutoff of 2. Results Twenty-three analytes were detected. All strains, except the F28 induced an increase in the production of 18, and a decrease in 5 cytokines/chemokines at 24, 48 and 72h post-infection, compared to the uninfected control. However, F28 induced an increased production of all 23 cytokines/chemokines at 48 and 72h. A gradual increase in anti-, pro-inflammatory cytokines and chemokines that peaked mostly at either 48 or 72h, was induced in a strain specific manner. Changes in host gene expression were observed in surface receptors, interleukins, chemokines, transcriptional factors, human leukocyte antigen and interferon associated proteins. High fold changes were observed for HLA (B, C, F and H) genes, TLR5 and NOD2 receptors. The Unique strain induced the lowest fold changes while the F15/LAM4/KZN and F11 strains induced the highest fold changes in most genes. Hierarchical clustering showed that the Unique strain clustered separately from the highly virulent ones. Conclusion Clinical strains of M. tuberculosis of different genotypes induce specific patterns of in vitro cytokine production and immune gene expression in pulmonary epithelial cells. Furthermore, our results support findings that lung epithelial cells are involved in antigen processing and presentation.