Intestinal inflammation is associated with enhanced mucosal hypoxia, which contributes to the ongoing inflammatory process and hampers appropriate mucosal healing. We questioned whether local treatment with an oxygen (O 2 )-carrying and -releasing molecule (oxygenated perfluorodecalin, O 2 -PFD) could positively influence the course of experimental colitis. The impact of intrarectal (IR) treatment with O 2 -PFD was tested using the murine dextran sodium sulfate (DSS)-induced model of distal colitis, both in preventive and therapeutic settings. Colonic mucosal hypoxia was visualized by pimonidazole staining. Colonic permeability was evaluated with FITC-dextran. In the preventive study, mice treated with O 2 -PFD were protected against DSS colitis compared with saline-treated mice, as demonstrated by reduced shortening of colon length, reduced colonic tumor necrosis factor-alpha levels and a lower histological inflammation score (Po0.05 for all parameters). In the therapeutic study, administration of O 2 -PFD resulted in accelerated recovery of colitis compared with saline-treated littermates, and this was reflected by a better weight evolution, lower myeloperoxidase activity and a lower histological inflammation score (Po0.05 for all parameters). It was found that O 2 -PFD established its therapeutic effects through (1) intrinsic anti-inflammatory effects of the PFD molecule and (2) O 2 -induced preservation and healing of the intestinal epithelial surface. Further in vitro and in vivo studies showed that the barrier-protective activity of O 2 -PFD was obtained through prevention of colonocyte apoptosis and stimulation of colonocyte proliferation during inflammatory hypoxia. These data show that IR treatment with O 2 -PFD promotes colitis healing by the combined actions of direct anti-inflammatory effects and O 2 -induced restitution of the epithelial barrier. As such, O 2 -PFD enemas could be an attractive treatment option for patients with distal inflammatory bowel disease.