Chemoresistance contributes to cancer relapse and increased mortality in a variety of cancer types, raising a pressing need to better understand the underlying mechanism. MUC1 is abnormally overexpressed in numerous carcinomas and associated with poor prognosis. However, the functional significance of MUC1 in chemoresistance has not been fully elucidated. Here, we showed that MUC1 expression was considerably induced in cells that had acquired chemoresistance at both transcriptional and post-translational levels. Using gain- and loss-of function approaches, we demonstrated a critical role of MUC1 in induction of drug resistance. Through stimulation of EGFR activation and nuclear translocation, MUC1 increased the expression of ATP-binding cassette transporter B1 (ABCB1). Remarkably, targeted suppression of EGFR or ABCB1 by both shRNAs and inhibitors effectively reversed chemoresistance. Moreover, co-administration of the inhibitors of MUC1-EGFR-ABCB1 with paclitaxel significantly blocked not only tumor growth but also relapse in xenograft mouse model. Our data collectively support a model in which MUC1 induces acquired chemotherapy resistance by upregulating ABCB1 in an EGFR-dependent manner, providing a novel molecular basis of using the EGFR inhibitor in MUC1-positive cancers to prevent chemotherapy resistance.