Docosahexaenoic acid (DHA) is a long-chain polyunsaturated fatty acid mainly found in fish oil. Although several studies have suggested that it can alleviate allergy symptoms, its mechanism of action remains to be elucidated. In the present study, we found that docosahexaenoyl ethanolamide (DHEA), a metabolite of DHA produced in the human body, exerts the anti-allergic activity in vitro and in vivo. DHEA suppressed degranulation of rat basophilic leukemia RBL-2H3 cells and bone marrow-derived mast cells in a dose-dependent manner without cytotoxicity. This occurred due to a decrease in Ca2+ influx, which is critical for mast cell degranulation. DHEA also suppressed IgE-mediated passive cutaneous anaphylaxis reaction in mice. In addition, DHEA was demonstrated to lessen an allergic symptom in a mouse model of pollinosis and to alter the production of IgE and cytokines secreted by splenocytes collected from the pollinosis mice. Taken together, this study indicates that DHEA is a promising anti-allergic agent as it inhibits mast cell degranulation and modulates other immune cells.