Background Ulcerative colitis (UC) is a chronic condition of intestine illness accompanied by some unknown etiology with different immune, genetic and environmental factors. Objectives The current study aimed to evaluate the antioxidant and anti-inflammatory effects of the Melilotus officinalis L. in the acetic acid induced UC in rats. Materials and Methods The plant aqueous extraction and high performance liquid chromatography and Ferric Reducing Antioxidant Power (FRAP) assay were performed on aqueous extract to identify its compounds and antioxidant activities. Also, 70 adult male rats were and UC was induced using 3% acetic acid solution. They received different daily doses of M. officinalis L. in two forms (orally, 500 and 1000 mg/kg) and gel extract (10% and 20%). On the 7th day, the colon tissues were examined regarding the macroscopic and histopathology lesions plus oxidative stress and compared to the positive and negative control groups. Results HPLC analysis revealed that five grams of the flower powder contained 9.7 mg gallic acid, 99 mg catechin, 21.9 mg caffeic acid, 0.86 mg chlorogenic acid, 1.13 mg quercetin, 548.9 mg cinnamic acid, 289 mg coumarin and 126 mg p-coumaric acid. The FRAP value of the extract was 2.91 ± 0.14 μM/g. There were significant differences between the group of rats which received the gel or aqueous extract of the flower compared to the negative control group using normal saline and the base gel and they had no significant differences with the positive control group using the Asacol, regarding the pathologic, malondialdehyde, and weight improvements. Conclusions It can be concluded that the M. officinalis L. extract can be used as an effective medicine to treat UC in animal model and also in human subjects.