Xanthine oxidase (XO) is one of the major enzymes to generate superoxide anion (O2−), that is frequently associated with various diseases involving reactive oxygen species (ROS). 4-Amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP) is a potent XO inhibitor showing therapeutic potential for oxidative inflammatory diseases. However its very poor aqueous solubility makes pharmaceutical application difficult. To overcome this drawback, we have successfully synthesized a water soluble polyethylene glycol (PEG) conjugate of AHPP (PEG–AHPP) that exhibited good water solubility, forming micelles in aqueous solution. In the present study, the in vivo pharmacokinetics of this PEG–AHPP was examined. Further its therapeutic potential was investigated in dextran sulfate sodium (DSS) induced mouse colitis model. Compared to parental AHPP, the plasma t1/2 of PEG–AHPP was increased remarkably from 3 h to 14 h, indicating macromolecular nature of AHPP in circulation. In the DSS induced colitis model, oral administration of 2% DSS in drinking water resulted in the progression of the colitis with diarrhea and hematochezia as well as shortening of the large bowel. Administration of PEG–AHPP intravenously (10 mg/kg) or orally (20 mg/kg) suppressed pathogenesis significantly; namely diarrhea was reduced markedly, and the length of large bowel returned to almost normal level. Pathological examination clearly revealed improvement of colonic ulcer or necrosis. Production of inflammatory cytokines, i.e., interleukin-6 and tumor necrosis factor (TNF)-α, was significantly increased in DSS-induced colitis mice. However, it was markedly suppressed by PEG–AHPP administration. Similar results were found when serum 8-hydroxydeoxyguanosine (8-OHdG) and thiobarbituric acid reactive substances (TBARS), that are the index of oxidative injury, were measured. PEG–AHPP thus may be a potential candidate drug for ROS-related diseases including inflammatory bowel disease.