A large body of emerging evidence has revealed the role of p38/MK2 and PI3K/Akt/GSK3β cascades in the orchestrating process of colitis. Rutin, a bioflavonoid present in many fruits and vegetables, has been recognized to offer therapeutic attributes in acute colitis. However, its role in chronic colitic condition has not yet been delineated in reference to p38/MK2 and PI3K/Akt/GSK3β signalling. The present investigation assessed the efficacy and underlying molecular mechanism of rutin in alleviating DSS-induced chronic colitis. The analysis of signalling pathways demonstrated the robust activation of PI3K/Akt/GSK3β/MAPKs/NF-κB and p38/MK2 in DSS-induced colitis in animals, which was efficiently alleviated following the rutin treatment. In silico studies indicated its target specificity with these pathways. Rutin administration markedly improved the disease activity score, colon length, goblet cell loss and compromised colon epithelial integrity in colitic mice. Decreased expression of oxi-inflammatory markers such as IgM, IgE, iNOS, ICAM-1, HO-1 and Th1/IL-10 cytokines ratios after treatment suggests its efficacy in regulating effector, regulatory and B cell homeostasis. Additionally, rutin demonstrated its role in restoring epithelial integrity by modulating the transcript levels of tight junction proteins, mucus-secreting proteins, epithelial cell proliferation and apoptosis. Treg expansion revealed that rutin supplementation also exhibits an immune regulatory potential and suppresses inflammatory aggravation mediated by adaptive immune responses. Overall, results indicate that the modulation of p38/MK2 and PI3K/Akt/GSK3β/NF-κB pathways by rutin represents a novel therapeutic approach in chronic colitis that help to curb dysregulated intestinal integrity, cytokine ratio and splenic Tregs.