CD133 has been described as a marker for cancer stem cells (CSCs) in colorectal cancer. Additionally, it has been reported that CSCs are resistant to chemoradiotherapy (CRT). After previously observing that CD133 mRNA levels were elevated after CRT in rectal cancer patients, we further investigated CD133 expression in colorectal cancer following CRT using immunohistochemistry. Forty patients with primary colorectal cancers and 50 patients with rectal cancer who had received preoperative CRT followed by surgery were selected. Sections of formalin-fixed, paraffin-embedded specimens were stained for CD133, CK20 and Ki-67. To clarify the change of CD133 protein after irradiation, CD133 protein levels were examined in radiation-treated human colon cancer cell line HT29. We found four distinct patterns of CD133 staining defined by CD133 expression in luminal surface, in intraluminal cells and in cytoplasm. In total, CD133 expression was detected in 27.5% of non-CRT and 70% of CRT specimens. The frequency of CD133 staining in CRT specimens was significantly higher than that of non-CRT specimens. Seven out of 50 CRT specimens exhibited cytoplasmic staining. These cells with cytoplasmic CD133 expression did not express CK20 or Ki-67. The ratio of histopathological responder in cases with CD133 expression in both luminal surface and cytoplasm was significantly lower than that without it (P<0.05). In vitro study showed that CD133 protein was increased in a radiation-dose dependent manner. Further studies clarifying the role of CD133 in tumor re-growth and resistance to conventional CRT in colorectal cancer may assist the development of future cancer therapeutics.