The aim of this study is to explore the expression of CD109 in breast cancer stem cells and the relationship between CD109 protein and clinicopathological characteristics of breast cancer. CD44+/CD24- tumor cells (CSCs) were selected by flow cytometry. The protein expression of CD109 was analyzed by immunohistochemistry staining, and the relationship between CD109 and clinicopathological parameters of breast cancer was determined. CD109 positively regulated the proliferation of breast CSCs in vitro, and CD109 protein expression was significantly higher in triple-negative breast cancer (TNBC) compared to non-TNBC (63.78 vs. 3.71 %, P = 0.001). Moreover, CD109 protein expression was related to the histological grade of breast cancer (P = 0.015), whereas age (P = 0.731), tumor size (P = 0.995), clinical stage (P = 0.644), and lymph node metastasis (P = 0.924) were not. In the logistic regression model, histological grade (P = 0.001) and molecular type (P = 0.001) were significantly related to CD109 expression. The patients with high expression of CD109 protein had significantly poorer postoperative disease-specific survival than those with no or low expression of CD109 protein (P = 0.001). In the Cox regression, CD109 was an independent prognostic factor (P = 0.001). CD109 is highly expressed in TNBC and is a potential biomarker for the initiation, progression, and differentiation of breast cancer tumors.