Assessment of digestion colonic flora lactulose can be induced to produce hydrogen and prove lactulose with a potential anti-inflammatory effects. We established a mouse model of human ulcerative colitis (UC) by supplying mice with water containing dextran sulfate sodium (DSS). At 7-day, DSS-induced pathogenic outcomes, including loss of body weight, increase of colitis score, pathogenic shortening of colon length, elevated level of TNF-α, IL-1β, malondialdehyde (MDA) and myeloperoxidase (MPO) deficiency in colon lesions, were significantly suppressed by lactulose administration and intraperitoneally injected hydrogen water. After eliminating colon microflora by antibiotics, the anti-inflammatory effects of lactulose were significantly compromised. Histological analysis also revealed that the DSS mediated colonic tissue destruction accompanied by neutrophils infiltration was remarkably suppressed by lactulose and hydrogen water. The present study indicated that lactulose can prevent the development of DSS-induced colitis and alleviate oxidative stress state in colitis.