AMP-activated protein kinase (AMPK), a cellular energy sensor, has been reported to participate in modulating inflammatory responses, but its role in intestinal inflammation remains unclear. IBD has been characterized by excessive innate and adaptive immune responses. Here, the roles of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an agonist of AMPK, in regulating immune responses of experimental colitis were investigated. In vitro effects of AICAR on LPS-induced macrophage activation and Th1 and Th17 differentiation, as well as in vivo effects of AICAR in mice with 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis, were explored. In acute colitis, daily AICAR treatment commenced 2 days after TNBS delivery (day 1), while in relapsing colitis, AICAR treatment commenced after three weekly TNBS administrations. Colon inflammation, production of proinflammatory cytokines and NF-κB activation in colon tissues, and Th1 and Th17 cell populations in lamina propria mononuclear cells (LPMCs) and mesenteric lymph node cells (MLNs) were assayed. Results show that AICAR significantly inhibited in vitro LPS-induced macrophage activation and Th1 and Th17 cell differentiation. Administration of AICAR was therapeutically effective in ameliorating acute and relapsing experimental colitis, as shown by reduced body weight loss and significant attenuation in colon histological inflammation. Moreover, this treatment inhibited NF-κB activation in macrophages, and reduced levels of TNF, Th1- and Th17-type cytokines, and Th1 and Th17 cell populations in LPMCs and MLNs. AICAR-initiated AMPK activation may act as a central downregulator in ongoing innate and adaptive immune responses of murine colitis, providing a novel therapeutic approach in the treatment of IBD.