Indirubin and isatin have been used in the treatment of inflammatory diseases due to their anti-inflammatory properties. This study aimed to evaluate the combined effect of indirubin and isatin on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC). UC was induced by the administration of 3% (w/v) DSS solution, and then the model mice were administered indirubin (10 mg/kg body mass) and (or) isatin (10 mg/kg body mass) by gavage once daily for 7 days. The results showed that indirubin and isatin, individually or combined, significantly inhibited weight loss, lowered disease activity index (DAI), ameliorated pathological changes, decreased the levels of pro-inflammatory mediators and myeloperoxidase (MPO) activity, increased the expression of anti-inflammatory cytokines and Foxp3, suppressed CD4 T cell infiltration, and inhibited oxidative stress and epithelial cell apoptosis. Additionally, indirubin and isatin, both individually and combined, can also inhibit activation of the NF-κB and MAPK pathways induced by DSS. The protective effect of combination therapy against UC was superior to that of single-agent treatment. These results suggest that indirubin combined with isatin attenuates DSS-induced UC, and changes to the NF-κB and MAPK signaling pathways may mediate the protective effects of indirubin and isatin in UC.