The incidence of inflammatory bowel disease (IBD), a chronic inflammatory condition of the gastrointestinal tract characterized by a progressive and unpredictable disease course, has been increasing in many parts of the world. Inflammation and tight junction disruption in the mucosa play key roles in IBD pathogenesis, highlighting inflammation inhibition as a promising therapeutic strategy. Betanin, a red beetroot pigment, possesses potent anti-inflammatory and antioxidant properties, suggesting its potential as a treatment to ameliorate for IBD symptoms. In this study, we evaluated whether betanin alleviates symptoms in a mouse model of colitis and explored its underlying mechanisms. Betanin administration via intraperitoneal or oral injection reduced body weight loss and rectal bleeding and alleviated histological damage in the distal colon of dextran sulfate sodium (DSS)-induced IBD mice model. Moreover, intraperitoneal injection of betanin inhibited the activation of macrophages and enteric glial cells in DSS-induced colitis mice model. Furthermore, it attenuated DSS-induced downregulation of the tight junction proteins occludin and claudin-1. These findings suggest that betanin may improve IBD symptoms by suppressing inflammation and preserving tight junction function in colitis mice model.