Accumulated data have shown that alternatively activated macrophage exerts a modulatory role in many diseases, including colitis. Interleukin-33 (IL-33), a critical modulator in adaptive and innate immune, has been implicated in autoimmunity and inflammation. Previously, we have reported that IL-33 functions as a protective modulator in TNBS-induced colitis, which is closely related to a Th1-to-Th2/Treg switch. Here, we present novel evidence suggesting that IL-33 primes macrophage into alternatively activated macrophages (AAM) in TNBS-induced colitis. The strong polarized effect of IL-33 was tightly associated with the markedly increased induction of Th2-type cytokines. To confirm the beneficial effects of AAM induced by IL-33, peritoneal AAMs isolated from IL-33-treated mice were transferred to recipient mice with TNBS colitis. The adoptive transfer resulted in prominent inhibition of disease activity and inflammatory cytokines in the TNBS-treated mice. In conclusion, our data provide clear evidence that IL-33 plays a protective role in TNBS-induced colitis, which is closely related to AAM polarization.