Inflammatory bowel disease (IBD) is a chronic and relapsing inflammatory disorder of the gastrointestinal tract (GI). Currently, the treatment options for IBD are limited. It has been reported that a novel bioactive mitochondrial-derived peptide (MOTS-c) encoded in the mitochondrial 12S rRNA, suppresses inflammatory response by enhancing the phagocytosis of macrophages. The aim of this study was to investigate the protective effects of MOTS-c against dextran sulfate sodium (DSS)-induced colitis. The results showed that intraperitoneal (i.p.) administration of MOTS-c significantly ameliorated the symptoms of DSS-induced experimental colitis, such as body weight loss, colon length shortening, diarrhea, and histological damage. MOTS-c down-regulated the expression of pro-inflammatory cytokines, decreased the plasma levels of myeloperoxidase, and inhibited the activation of macrophages and recruitment of neutrophils. Moreover, treatment with MOTS-c exhibited anti-apoptotic effects and significantly suppressed the phosphorylation of AMPKα1/2, ERK, and JNK. Notably, oral administration of MOTS-c did not result in any significant improvements. Screening of cell penetrating peptides was performed, (PRR)5 was linked to the C-terminus of MOTS-c through a linker to synthesize a new molecule (termed MP) with better penetration into the colon epithelium. In vitro experiments revealed the longer half-life of MP than MOTS-c, and in vivo experiments showed that oral administration of MP significantly ameliorated DSS-induced colitis. CONCLUSION: The present results demonstrate a protective role of MOTS-c in experimental IBD.