The heavy metal lead (Pb) is a toxic contaminant that induces a range of adverse effects in humans. The present study demonstrated for the first time that dietary supplementation with a galactooligosaccharide (GOS) promotes fecal Pb excretion and reduces Pb accumulation in the blood and tissues of mice. The effects against Pb exposure were also observed in mice that received the fecal microbiota from donors treated with GOS, but were diminished in gut microbiota-depleted mice that received antibiotic pre-treatment, indicating that the protection by GOS administration was dependent on the modulation of the gut microbiota. We also provide evidence that the protective mechanism of GOS supplementation was related to the enhanced abundance of intestinal bacteria with good Pb-binding ability, recovery of the gut barrier function, modulation of bile acid metabolism, and improved essential metal utilization. These results indicate that GOS can be considered a potentially protective prebiotic against Pb toxicity.