Radiation-induced intestinal fibrosis (RIF) is a serious complication after abdominal radiotherapy. We show RIF is mediated by eosinophil interactions with a-smooth muscle actin (a-SMA)+ stromal cells. Abdominal irradiation induced fibrosis of the submucosa (SM) associated with the excessive accumulation and degranulation of eosinophils in the absence of lymphocytes. Eosinophil-deficiency markedly ameliorated RIF, suggesting their importance. Chronic crypt necrosis post-irradiation elevated extracellular adenosine triphosphate levels, which induced C-C motif chemokine 11 (CCL11) and granulocyte-macrophage colony-stimulating factor (GM-CSF) expression by pericryptal a-SMA+ cells that attracted and activated eosinophils, respectively. Transforming growth factor-b1 from GM-CSF-stimulated eosinophils promoted collagen expression by a-SMA+ cells. Upon co-stimulation with GM-CSF and CCL11, eosinophils released granule protein, which up-regulated CCL11 and profibrotic matrix metalloproteinase expression by a-SMA+ cells, facilitating eosinophil-mediated fibrogenesis. Thus, the mutual activation of eosinophils and a-SMA+ cells creates a positive feedback loop that mediates RIF progression. These findings aid the development of effective therapeutic strategies.