Regulatory B cells (Breg) have attracted increasing attention for their roles in maintaining peripheral tolerance. Interleukin 33 (IL-33) is a recently identified IL-1 family member, which leads a double-life with both pro- and anti-inflammatory properties. We report here that peritoneal injection of IL-33 exacerbated inflammatory bowel disease in IL-10-deficient (IL-10^−/−) mice, whereas IL-33-treated IL-10-sufficient (wild type) mice were protected from the disease induction. A phenotypically unconventional subset(s) (CD19⁺CD25⁺CD1d^hiIgM^hiCD5^-CD23^-Tim-1^-) of IL-10 producing Breg-like cells (Breg^IL-33) was identified responsible for the protection. We demonstrated further that Breg^IL-33 isolated from these mice could suppress immune effector cell expansion and functions and, upon adoptive transfer, effectively blocked the development of spontaneous colitis in IL-10^−/− mice. Our findings indicate an essential protective role, hence therapeutic potential, of Breg^IL-33 against mucosal inflammatory disorders in the gut.