Bacterial infection is the main cause of acute colitis with Salmonella typhimurium being a common culprit. However, there is a lack of research on the safe and efficient therapy for S. typhimurium-induced colitis. Sublancin, an antimicrobial peptide (AMP), is supposed to have strong antibacterial activity and low susceptibility to resistance. Therefore, this study aimed to investigate the effect of sublancin on S. typhimurium-induced colitis. An acute colitis model was established by S. typhimurium infection in mice. The therapy was administered by oral gavage. The trial was divided into four groups: control (Con), infection (Inf), low concentration of sublancin (L-Sub) and high concentration of sublancin (H-Sub). After 3 days of treatment, H-Sub group exhibited ameliorated body weight loss and improved colon tissue morphology compared to Inf group. Moreover, there was a notable decrease in the inflammatory response and an alleviation of the intestinal epithelial barrier damage in the colons of H-Sub group compared to Inf group. Consistently, high-dose sublancin also reduced the weight and bacterial load of spleen and liver after S. typhimurium attack. Mechanically, gut microbiota analysis performed by 16 S rRNA gene seq revealed that sublancin balanced microbial dysbiosis in colonic content, suggesting that increased Paramuribaculum, Clostridium_AQ and decreased Muribaculum may response to sublancin. These findings may help providing a new therapy of AMP in bacterial colitis.