In regions with a high infectious disease burden, concerns have been raised about the safety of iron supplementation because higher iron concentrations in the gut lumen may increase risk of enteropathogen infection. The aim of this study was to investigate interactions of the enteropathogen Salmonella enterica ssp. enterica Typhimurium with intestinal cells under different iron concentrations encountered in the gut lumen during iron deficiency and supplementation using an in vitro colonic fermentation system inoculated with immobilized child gut microbiota combined with Caco-2/HT29-MTX co-culture monolayers. Colonic fermentation effluents obtained during normal, low (chelation by 2,2'-dipyridyl) and high iron (26.5 mg iron/L) fermentation conditions containing Salmonella or pure Salmonella cultures with similar iron conditions were applied to cellular monolayers. Salmonella adhesion and invasion capacity, cellular integrity and immune response were assessed. Under high iron conditions in pure culture, Salmonella adhesion was 8-fold increased compared to normal iron conditions while invasion was not affected leading to decreased invasion efficiency (-86%). Moreover, cellular cytokines IL-1β, IL-6, IL-8 and TNF-α secretion as well as NF-κB activation in THP-1 cells were attenuated under high iron conditions. Low iron conditions in pure culture increased Salmonella invasion correlating with an increase in IL-8 release. In fermentation effluents, Salmonella adhesion was 12-fold and invasion was 428-fold reduced compared to pure culture. Salmonella in high iron fermentation effluents had decreased invasion efficiency (-77.1%) and cellular TNF-α release compared to normal iron effluent. The presence of commensal microbiota and bacterial metabolites in fermentation effluents reduced adhesion and invasion of Salmonella compared to pure culture highlighting the importance of the gut microbiota as a barrier during pathogen invasion. High iron concentrations as encountered in the gut lumen during iron supplementation attenuated Salmonella invasion efficiency and cellular immune response suggesting that high iron concentrations alone may not lead to an increased Salmonella invasion.