Astragalus membranaceus is a medicinal herb with potential immunomodulatory property, which has been used in treating colitis-related diarrhea. In the present investigation, we aimed to further explore its anti-inflammatory activity by studying the immunoregulatory mechanism of Astragalus root extract (Am) through different routes of administration in hapten-induced colitis. 2,4-Dinitrobenzene sulfonic acid (DNBS) was used to induce experimental colitis in Sprague-Dawley rats. Results have indicated that both oral and intracolonic Am treatments (administered twice daily for three consecutive days following colitis induction) exhibited significant protection against DNBS-induced colitis in rats, indicated by decreased colonic lesion area and histological damage score as well as amelioration of the elevated colonic myeloperoxidase activity. Western immunoblotting has revealed that oral Am could diminish the overexpression of tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta, while concomitantly abolishing the inhibition of IL-10 expression in rats' colon under colitis condition. On the other hand, intracolonic Am could only reduce TNF-alpha and interferon-gamma overexpression. In summary, we have demonstrated that both oral and locally administered Am possess protective effects against experimental colitis through differential modulation of colonic cytokines. This study provides important new insights that may contribute to further development of Am as a novel therapeutic agent for treating colitis diseases.