Mycobacterium avium: subsp. hominissuis (MAH) is an opportunistic pathogen that commonly infects immunocompromised individuals. Recently, we described an invasive phenotypic change MAH undergoes when incubated with lung airway epithelial host cells for 24 h, which is accompanied with microaggregate formation in vitro. The microaggregate phenotype also resulted in higher colonization in the lungs of mice early during infection. Previously, we identified genes highly regulated during microaggregate formation and further characterized the function of two highly upregulated bacterial proteins, mycobacterial binding protein-1 (MBP-1) and mycobacterial inversion protein-1 (MIP-1), which were found to be involved in binding and invasion of the respiratory mucosa. While these studies are valuable in understanding the pathogenesis of MAH, they primarily investigated the bacteria during microaggregate infection without commenting on the differences in the host response to microaggregate and planktonic infection. The bacteria-host interaction between microaggregates and epithelial cells was examined in a variety of assays. Using a transwell polarized epithelial cell model, microaggregates translocated through the monolayer more efficiently than planktonic bacteria at set timepoints. In addition, during infection with microaggregate and planktonic bacteria, host phosphorylated proteins were identified revealing differences in immune response, glutathione synthesis, and apoptosis. The host immune response was further investigated by measuring pro-inflammatory cytokine secretion during microaggregate and planktonic infection of BEAS-2B bronchial epithelial cells. The epithelial cells secreted more CCL5 during infection with microaggregates suggesting that this chemokine may play an important role during microaggregate invasion. Subsequent experiments showed that microaggregates are formed more efficiently in the presence of CCL5, suggesting that MAH had evolved a strategy to use the host response in its benefit. Collectively, this study establishes the different nature of infection by planktonic bacteria and microaggregates.