Human leukocyte antigen-G (HLA-G) is known to be implicated in a tumor-driven immune escape mechanism in malignancies. The purpose of this study was to investigate HLA-G polymorphism and expression in breast cancer. HLA-G alleles were determined by direct DNA sequencing procedures from blood samples of 80 breast cancer patients and 80 healthy controls. Soluble HLA-G (sHLA-G) was measured by enzyme-linked immunosorbent assay (ELISA) from serum specimens. HLA-G expression in breast cancer lesions was also analyzed by immunohistochemistry staining. The presence of HLA-G 3' untranslated region (UTR) 14-bp sequence was analyzed and found to be associated with reduced risk of breast cancer susceptibility based on HLA-G expression in tissues (P = 0.0407). Levels of sHLA-G were higher in the breast cancer group (median 117.2 U/mL) compared to the control group (median 10.1 U/mL, P<0.001). The area under the receiver operating characteristic curve (AU-ROC) values of sHLA-G for differentiating breast cancer from normal controls and for detecting metastasis from other stages of breast cancer were 0.89 and 0.79, respectively. HLA-G polymorphism and expression may be involved in breast carcinogenesis and sHLA-G concentrations could be used as a diagnostic marker for detecting breast cancer.