Ulcerative colitis (UC) is a kind of inflammatory bowel disease (IBD) that often recurs and is difficult to cure, and no drugs with few side effects are available to treat this disease. LfcinB is a small molecular peptide obtained by the hydrolysis of bovine lactoferrin in the digestive tract of animals. It has strong antibacterial and anti-inflammatory activities. However, direct evidence that LfcinB improves the condition of colitis in mice is rarely reported. In this study, UC was induced in mice by adding 2.5% dextran sulfate (DSS) to drinking water and LfcinB was orally administered. The results showed that oral administration of LfcinB improved colonic tissue damage and inflammatory cell infiltration, increased the expression of tight junction proteins, and down-regulated the phosphorylation of proteins related to the NF-κB/MAPK inflammatory signalling pathway in mice. It also significantly suppressed the relative abundance of potentially pathogenic bacteria (Bacteroides, Barnesiella and Escherichia) in the intestinal flora. In conclusion, oral administration of LfcinB significantly alleviated DSS-induced UC. This may be related to the regulation of inflammatory signalling pathways and gut microbial composition by LfcinB.