Metabolic syndrome (MetS, i.e., type 2 diabetes and obesity) is often associated with dysbiosis, inflammation, and leaky gut syndrome, which increase the content of oxygen and reactive oxygen species (ROS) in the gastrointestinal (GI) tract. Using near-infrared fluorescent, in situ imaging of ROS, we evaluated the effects of oral administration of elemental iron powder (Fe) on luminal ROS in the GI tract and related these changes to glucose metabolism and the gut microbiome. C57Bl/6J mice fed low-fat or high-fat diets and gavaged with Fe (2.5 g per kg), in both single- and repeat-doses, demonstrated decreased levels of luminal ROS. Fourteen days of repeated Fe administration reduced hyperglycemia and improved glucose tolerance in the obese and hyperglycemic animals compared to the untreated obese controls and reduced the relative amount of iron oxides in the feces, which indicated an increased redox environment of the GI tract. We determined that Fe administration can also be used as a diagnostic assay to assess the GI microenvironment. Improved metabolic outcomes and decreased gastrointestinal ROS in Fe-treated, high-fat diet-fed animals correlated with the increase in a co-abundance group of beneficial bacteria, including Lactobacillus, and the suppression of detrimental populations, including Oscillibacter, Peptococcus, and Intestinimonas. Daily Fe treatment also increased the relative abundance of amplicon sequence variants that lacked functional enzymatic antioxidant systems, which is consistent with the ability of Fe to scavenge ROS and oxygen in the GI, thus favoring the growth of oxygen-sensitive bacteria. These findings delineate a functional role for antioxidants in modification of the GI microenvironment and subsequent reversal of metabolic dysfunction.