Alopecia is known as a symptom of acute radiation, yet little is known concerning the mechanism of this phenomenon and the alteration of hair protein profiles. To examine this, 6-week-old male C57/BL6 mice were exposed to 6 Gy of X-ray irradiation, which caused acute alopecia. Their hair and skin were collected, and hair proteins were analyzed with liquid chromatography/electrospray-ionization mass spectrometry and immunohistochemistry. No change was observed in the composition of major hair keratins, such as Krt81, Krt83 and Krt86. However, cytokeratin Krt15 and CD34, which are known as hair follicle stem cell markers, were decreased in alopecic mice. Cytokeratin Krt5, which is known as a marker for basal and undifferentiated keratinocytes, was increased in the epidermis of alopecic mice. These findings suggest that radiation damages hair stem cells and the differentiation of keratinocytes in the epidermis. For the evaluation of radiation exposure, chromosomal aberration is considered to be the gold standard, yet our results suggest that Krt5 may be a novel biological marker for acute radiation symptoms.