Y-box-binding protein 1 (YB-1) has been identified as a pleotropic oncoprotein in multiple human malignancies and is involved in the regulation of various DNA/RNA-dependent events, including transcription and translation. However, little is known about expression pattern and underlying functions of YB-1 in esophageal squamous cell carcinoma (ESCC). In this study, we report that up-regulated YB-1 is closely correlated with TNM stage and tumor size in ESCC specimens and predicts a poor prognosis in ESCC patients. Genetic silencing of YB-1 inhibits cell proliferation and invasive potential by regulating its targeted genes associated with tumor growth and metastasis. In particular, we also show that silencing of YB-1 enhances chemotherapy-induced cytotoxicity in EC109 and TE-1 cells by targeting multidrug resistance 1 (MDR1). Our findings demonstrate the oncogenic roles of YB-1 in ESCC and support it as a target for ESCC therapy.