The skin-associated chemokine CCL27 and its receptor CCR10 mediate the immune response of skin-homing T cells. The CCL27 secreted from keratinocytes was reportedly involved in inflammatory skin diseases such as atopic dermatitis, contact dermatitis, and psoriasis. However, whether ionizing radiation increases the levels of CCL27 secretion still remains unclear. In HaCaT cells, a human keratinocyte cell line, CCL27 secretion was markedly increased after X-ray irradiation. We further found that irradiation boosted the generation of reactive oxygen species (ROS), which was concomitant with the release of tumor necrosis factor-alpha (TNF-alpha). Moreover, alteration of ROS in irradiated HaCaT cells correlated with TNF-alpha secretion, indicating a positive loop of TNF-alpha secretion and ROS generation. This positive loop regulated the secretion of CCL27 from irradiated cells. We therefore concluded that the cross talk between TNF-alpha and ROS after keratinocytes was exposed to radiation, triggered CCL27 secretion for subsequent inflammation response.