Background: Skin and oral mucosa are continuously exposed to potential metal sensitizers while hosting abundant microbes, which may influence the host response to sensitizers. This host response may also be influenced by the route of exposure that is skin or oral mucosa, due to their different immune properties. Objective: Determine how commensal Streptococcus mitis influences the host response to nickel sulfate (sensitizer) and titanium(IV) bis(ammonium lactato)dihydroxide (questionable sensitizer) in reconstructed human skin (RHS) and gingiva (RHG). Methods: RHS/RHG was exposed to nickel or titanium, in the presence or absence of S. mitis for 24 hours. Histology, cytokine secretion, and Toll-like receptors (TLRs) expression were assessed. Results: S. mitis increased interleukin (IL)-6, CXCL8, CCL2, CCL5, and CCL20 secretion in RHS but not in RHG; co-application with nickel further increased cytokine secretion. In contrast, titanium suppressed S. mitis–induced cytokine secretion in RHS and had no influence on RHG. S. mitis and metals differentially regulated TLR1 and TLR4 in RHS, and predominantly TLR4 in RHG. Conclusion: Co-exposure of S. mitis and nickel resulted in a more potent innate immune response in RHS than in RHG, whereas titanium remained inert. These results indicate the important influence of commensal microbes and the route of exposure on the host's response to metals.