Radiation-induced acute skin injury and consequent fibrosis are common complications of cancer radiotherapy and radiation accidents. Stromal cell-derived factor-1α (SDF-1α) and its receptor, CXC chemokine receptor 4 (CXCR4) have been shown to be involved in multiple cellular events. However, the role of SDF-1α/CXCR4 axis in radiation-induced acute injury and fibrosis of skin has not been reported. In this study, we found that the expression of SDF-1α and CXCR4 was significantly increased in irradiated skin tissues of humans, monkeys and rats, compared to their nonirradiated counterparts. Mice with keratinocyte-specific ablation of CXCR4 showed less severe skin damage than wild-type mice after receiving a 35 Gy dose of radiation. Consistently, subcutaneous injection of AMD3100, an FDA approved SDF-1α/CXCR4 inhibitor, attenuated skin injury and fibrosis induced by exposure to radiation in a rat model. Mechanically, the SDF-1α/CXCR4 axis promotes pro-fibrotic TGF-b/Smad signaling through the PI3K-MAPK signaling cascade in human keratinocyte HaCaT cells and skin fibroblast WS1 cells. AMD3100 inhibited Smad2 nuclear translocation and transcriptional activity of Smad2/3 induced by radiation, which suppressed the pro-fibrotic TGF-b/Smad signaling pathway activated by exposure. Taken together, these findings demonstrate the involvement of SDF-1α/CXCR4 axis in radiation-induced acute injury and fibrosis of skin, and indicate that AMD3100 would be an effective countermeasure against these diseases.