Radiotherapy for lymphoma, gynecologic cancers, and sarcomas of the upper abdomen may result in radiation-induced kidney injury. Moreover, it is possible that radiation nephropathy could occur after a nuclear accident. Radiation nephropathy usually progresses very slowly, over a period of several years. The precise pathogenesis and/or mediators involved in radiation nephropathy remain under active investigation. It has been indicated that ionizing radiation induces senescence, which is one of the hallmarks of aging. As the senescent cell fraction following irradiation is known to increase, we investigated whether ionizing radiation causes the renal injury by inducing cellular senescence. In the present study, we evaluated the effects of ionizing radiation on canine renal epithelial cells (MDCK), which predominantly used in in vitro renal cell models. We found that the exposure of ionizing radiation on MDCK cells induced cellular senescence. Notably, cleaved form of Klotho, a renal neuraminidase that is essential for the function of fibroblast growth factor 23, was significantly increased following irradiation in culture media of MDCK cells. Lectin affinity analysis determined that these secreted klotho reduced the sialylated form of epithelial calcium channel (TRPV5), in MDCK cells. Furthermore, Klotho expression was decreased in renal tissues of radiation-exposed mice. Collectively, our data suggest that increase of secreted klotho and their neuraminidase activity to TRPV5 following irradiation may contribute to the development of renal dysfunction through the acceleration of cellular senescence.