Iron (Fe) deficiency affects an estimated 2 billion people worldwide, and Fe supplements are a common corrective strategy. The impact of Fe deficiency and Fe supplementation on the complex microbial community of the child gut was studied using in vitro colonic fermentation models inoculated with immobilized fecal microbiota. Chyme media (all Fe chelated by 2,2′-dipyridyl to 26.5 mg Fe L−1) mimicking Fe deficiency and supplementation were continuously fermented. Fermentation effluent samples were analyzed daily on the microbial composition and metabolites by quantitative PCR, 16S rRNA gene 454-pyrosequencing, and HPLC. Low Fe conditions (1.56 mg Fe L−1) significantly decreased acetate concentrations, and subsequent Fe supplementation (26.5 mg Fe L−1) restored acetate production. High Fe following normal Fe conditions had no impact on the gut microbiota composition and metabolic activity. During very low Fe conditions (0.9 mg Fe L−1 or Fe chelated by 2,2′-dipyridyl), a decrease in Roseburia spp./Eubacterium rectale, Clostridium Cluster IV members and Bacteroides spp. was observed, while Lactobacillus spp. and Enterobacteriaceae increased consistent with a decrease in butyrate (−84%) and propionate (−55%). The strong dysbiosis of the gut microbiota together with decrease in main gut microbiota metabolites observed with very low iron conditions could weaken the barrier effect of the microbiota and negatively impact gut health.