Protein reversionless 3-like (REV3L), the catalytic subunit of DNA polymerase (pol) ζ, is well known to participate in error-prone translesion synthesis (TLS) with less stringent and lower processivity. Recent evidence has demonstrated that REV3L is involved in carcinogenesis and tumor progression. However, the function of REV3L remains unclear in esophageal squamous cell carcinoma (ESCC). In the present study, we examined REV3L expression in ESCC tissues and its association with clinicopathological parameters. REV3L was found to be significantly upregulated and correlated with lymph node metastasis and clinical stage in the ESCC tissues. To further investigate the potential role of REV3L in esophageal cancer, stable ESCC cell lines with suppression of REV3L expression were established. Downregulation of REV3L expression led to a decrease in cell proliferation and invasive capacity partly through suppression of cyclin D1 and survivin expression, and an increase in cellular sensitivity to 5-fluorouracil (5-FU) by induction of G1 phase arrest and apoptosis. Therefore, REV3L plays an important role in ESCC progression and chemoresistance, and is a potential diagnostic marker and therapeutic target for ESCC.