Abstract Background Nasopharyngeal colonization by Streptococcus pneumoniae is characterized by bacterial adherence to epithelial cells, microinvasion, and innate immune activation. Previously, we have shown that two serotype 6B S pneumoniae mutant strains affecting bacterial metabolism (ΔproABC/pia and Δfhs/pia) colonize humans and mice, but in a murine disease model do not cause invasive infection. Methods Using an experimental human pneumococcal challenge model, ex vivo airway cells, and in vitro nasopharyngeal epithelium, we explore whether microinvasion and innate immune responses persist despite disease attenuation. Results We show that under serum stress, these biosynthesis gene mutations had a broad but different impact on pneumococcal virulence gene expression, oxidative stress regulation, and purine and carbohydrate metabolism genes. However, although these mutations did not attenuate microinvasion in human challenge and epithelial models, there was less transmigration of Detroit 562 nasopharyngeal epithelial cells by the mutants compared to wild-type. Cellular reorganization of primary human airway epithelium varied considerably between strains. Compared to wild-type, infection of Detroit 562 epithelial cells by the Δfhs/piaA strain, but not the ΔproABC/piaA strain, was less proinflammatory, induced less caspase 8 production, and was associated with increased pneumococcal hydrogen peroxide and reduced pneumolysin secretion. Conclusions These findings suggest that differences in microinvasion and epithelial responses were driven by the differential expression of multiple bacterial virulence and metabolic pathways. These data highlight the complex impact of single gene mutations on bacterial virulence and suggest that the virulence determinants of pneumococcal epithelial colonization, microinvasion, and innate immunity are not necessarily directly linked to disease.