AIM: To study the effect of circulating cell-free oxyhemoglobin (FHb) on intestinal microcirculation and intestinal epithelial injury in a rat model. METHODS: To induce elevated intravascular circulating FHb, male Sprague-Dawley rats received water or FHb infusion. Microcirculatory changes in jejunum, ileum and colon were evaluated using fluorescent microspheres. Intestinal injury was quantified as plasmatic release of ileal lipid binding protein (iLBP) and verified by histological analysis of the ileum. RESULTS: Water and FHb infusions resulted, when compared with saline infusion, in reduced intestinal microcirculation (after 30 min P < 0.05, or better; after 60 min FHb infusion P < 0.05 for jejunum and colon). Circulating FHb levels correlated significantly with release of iLBP (Spearman r = 0.72, P = 0.0011). Epithelial cell injury of the villi was histologically observed after water and FHb infusions. CONCLUSION: This study shows that circulating FHb leads to a reduction in intestinal microcirculatory blood flow with marked injury to intestinal epithelial cells. These data support the hypothesis that circulating FHb contributes to the development of intestinal injury.