Acyl-CoA binding domain containing 3 (ACBD3) is involved in the maintenance of Golgi structure and function through its interaction with the integral membrane protein. However, the clinical significance and biological role of ACBD3 in breast cancer remain unclear. Herein, we found that the mRNA and protein levels of ACBD3 were markedly up-regulated in breast cancer cells and tissues. Immunohistochemical analysis of breast cancer tissues demonstrated that ACBD3 overexpression was significantly associated with advanced clinicopathological features. Univariate and multivariate analysis indicated that ACBD3 overexpression correlates with poor prognosis in breast cancer. Furthermore, overexpressing ACBD3 promoted, while silencing ACBD3 inhibited, self-renewal and tumorigenesis in breast cancer cells in vitro and in vivo respectively. Importantly, upregulating ACBD3 promoted the self-renewal and tumorigenesis of breast cancer cells via activating the Wnt/beta-catenin signaling, and the pro-self-renewal effect of ACBD3 in breast cancer was antagonized by the Wnt signaling inhibitor TCF4-siRNA and Lef1-siRNA.These findings indicate that ACBD3 may represent candidate therapeutic targets to enable the elimination of breast cancer stem cells, providing the preclinical proof-of-concept for the prevention and treatment of breast cancer.