BLT2 is a low‐affinity leukotriene B₄ (LTB₄) receptor that is activated by 12(S)‐hydroxyhep‐tadeca‐5Z,8E,10E‐trienoic acid (12‐HHT) and LTB₄. Despite the well‐defined proinflammatory roles of BLT1, the in vivo functions of BLT2 remain elusive. To clarify the role of BLT receptors in intestinal inflammation, we assessed susceptibility to dextran sodium sulfate (DSS)‐induced colitis in mice lacking either BLT1 or BLT2. BLT2^–/– mice exhibited increased sensitivity to DSS as compared to wild‐type and BLT1^_/_ mice, with more severe body weight loss and inflammation. Expression of inflammatory cytokines such as interferon (IFN)‐γ, interleukin (IL)‐1β, and IL‐6, chemokines such as CXC chemokine ligand 9 (CXCL9) and C‐C motif chemokine 19 (CCL19), and metalloproteinases was highly up‐regulated in the colons of DSS‐treated BLT2^–/– mice, and there was an enhanced accumulation of activated macrophages. Phosphorylation of the signal transducer and activator of transcription 3 (STAT3) was also markedly accelerated in the crypts of DSS‐treated BLT2^_/_ mice. Madin‐Darby canine kidney II (MDCKII) cells transfected with BLT2 exhibited enhanced barrier function as measured by transepithelial electrical resistance (TER) and FITC‐dextran leakage through MDCK monolayers. Thus, BLT2 is expressed in colon cryptic cells and appears to protect against DSS‐induced colitis, possibly by enhancing barrier function in epithelial cells of the colon. These novel results suggest a direct anti‐inflammatory role of BLT2 that is distinct from the proinflammatory roles of BLT1.—Iizuka, Y., Okuno, T., Saeki, K., Uozaki, H., Okada, S., Misaka, T., Sato, T., Toh, H., Fukayama, M., Takeda, N., Kita, Y., Shimizu, T., Nakamura, M., Yokomizo, T. Protective role of the leukotriene B₄ receptor BLT2 in murine inflammatory colitis. FASEB J. 24, 4678–4690 (2010). www.fasebj.org