CXC Chemokine receptor type 4 (CXCR4) is commonly considered a potential marker for cancer stem cells (CSCs). Dedifferentiated-type chordoma (DTC) cells derived from a patient with recurrent chordoma exhibit high CXCR4 expression and demonstrate increased resistance to chemotherapeutic drugs and ionizing radiation (IR) compared to the conventional-type chordoma cell line, U-CH1. However, the precise role of CXCR4 in the stemness and IR resistance of DTC remains unclear. Therefore, this study aims to elucidate the correlation between the expression of CXCR4 and stemness and radioresistance in chordoma. DTC cells expressing CXCR4 (CXCR4+ DTC cells), isolated by magnetic-activated cell sorting, exhibited increased self-renewal activity, tumorigenicity, and IR resistance, accompanied by elevated Sox2 expression. Knockdown of CXCR4 expression using short hairpin RNA, inhibition of CXCR4 signaling with AMD3100, and targeting of STAT3, a downstream effector of CXCR4, with WP1066 in DTC cells significantly diminished their self-renewal ability, tumorigenic potential, IR resistance, and Sox2 expression. Additionally, transfection with a small interfering Sox2 RNA suppressed self-renewal activity, tumorigenicity, and IR resistance in DTC cells, whereas overexpression of CXCR4 reversed these effects in U-CH1 cells. Furthermore, DTC cells infected with shCXCR4 exhibited substantial tumor suppression, and the combination of IR and AMD3100 significantly reduced DTC tumor growth in a mouse xenograft model. These findings underscore the functional significance of CXCR4 as a CSC marker, highlighting its potential as a therapeutic target for malignant chordomas.