Ulcerative colitis (UC) is an inflammatory bowel disease, which is a chronic gastrointestinal disorder. Oldenlandia diffusa (OD) has been used as a traditional oriental medicine for inflammation. However, the regulatory effect and molecular mechanism of OD in intestinal inflammation are not yet understood. This study investigated the protective effect of OD in dextran sulfate sodium (DSS)-induced colitis. Mice treated with DSS showed remarkable clinical signs, including weight loss, and reduced colon length. Administration of OD attenuated these signs and significantly suppressed levels of interleukin (IL)-6, IL-1β and expression of cyclooxygenase-2 in DSS-treated colon tissues. OD also reduced the activation of transcription nuclear factor-κB p65 in DSS-treated colon tissues. Hentriacontane, a constituent of OD, attenuated weight loss, colon shortening, and levels of IL-6 caused by DSS. Taken together, the results provide experimental evidence that OD might be a useful therapeutic medicine for patients with UC.