Here, we examined how pneumococci adapt to the upper respiratory tract conditions, and how this affects interactions with the host. We established the intranasal microenvironment including metal ion and monosaccharide concentrations to create in vivo-mimicking medium to study pneumococcal adaptation to the human host. While growth of pneumococci in this medium was reduced, no major differences were found in capsular polysaccharide structure or thickness. Proteome analysis revealed a shift to galactose as carbohydrate source and decreased levels of fatty acid biosynthesis proteins and pneumolysin. Exo-metabolome analysis revealed accumulation of glycerophosphocholine leading to inhibited CRP and IgM binding to pneumococci. Pneumococci grown in in vivo-mimicking medium were more capable colonisers of differentiated primary epithelial cell layers and induced less epithelial cytokine release. Together these data show how pneumococci adapt to the nutrient-limited respiratory environment and modulate cells and humoral responses to facilitate persistent colonisation.