The Wnt/β-catenin pathway has important roles in chemoresistance and multidrug resistance 1 (MDR1) expression in some cancers, but its involvement in breast cancer and the underlying molecular mechanism are undefined. In this study, we demonstrated that the Wnt/β-catenin pathway is activated in chemoresistant breast cancer cells. Using a Wnt pathway-specific PCR array screening assay, we detected that Pygo2, a newly identified Wnt/β-catenin pathway component, was the most upregulated gene in the resistant cells. Additional experiments indicated that Pygo2 activated MDR1 expression in the resistant cells via the Wnt/β-catenin pathway. Moreover, the inhibition of Pygo2 expression restored the chemotherapeutic drug sensitivity of the resistant cells and reduced the breast cancer stem cell population in these cells in response to chemotherapy. Importantly, these activities induced by Pygo2 were mediated by MDR1. We also determined the effect of Pygo2 on the sensitivity of breast tumors resistant to doxorubicin in a mouse model. Finally, RNA samples from 64 paired patient tumors (before and after chemotherapy) highly and significantly overexpressed Pygo2 and/or MDR1 after treatment, thus underlining a pivotal role for the Pygo2-mediated Wnt/β-catenin pathway in the clinical chemoresistance of breast cancer. Our data represent the first implication of the Wnt/β-catenin pathway in breast cancer chemoresistance and identify potential new targets to treat the recurrence of breast cancer.