The plasma protein osteopontin (OPN) is considered to be a tumor biomarker, where elevated plasma levels are associated with poor prognosis. Additionally, OPN is expressed in the presence of tumor hypoxia, which is an adverse prognostic factor in radiation oncology. One of its receptors, the proposed tumor stem cell marker CD44, is also associated with aggressive tumors, shown for example in colon cancer. The expression of CD44 and its splice variants (particularly CD44v6) can be upregulated by OPN itself. In the present study, we aimed to investigate the influence of hypoxia on the expression of OPN and its binding partners CD44 and CD44v6 in colon carcinoma cell lines in vitro, using SW480, SW620, HT29 and HCT116 cells. Additionally, we investigated the effect of irradiation on the expression pattern of OPN and its ligands, and the influence of hypoxia on the clonogenic survival of the cells after irradiation. While the expression patterns were nearly unaltered by irradiation, hypoxia led to an upregulation of OPN protein expression and an increase in the radioresistance in all tested colorectal carcinoma cell lines. However, a similar clear statement with regard to the expression of CD44 and CD44v6 is not possible. We hypothesize that the OPN receptors differ in their expression pattern between cell lines depending on the degree of their malignancy.