Aberrant expression of miR-145 was associated with chemotherapy in multitype cancers. However, the underlying role and molecular mechanism of miR-145 in the sensitivity of esophageal squamous cell carcinoma (ESCC) to 5-FU remained largely unknown. Cell viability was determined by Cell Counting Kit-8 (CCK-8) assay. Gene expression levels were detected by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR). Protein expression levels were evaluated by Western blot. TargetScan was used for the prediction of binding sites for miRNA in mRNAs. The interaction between mRNA 3' UTR and miRNA was verified by dual luciferase reporter assay. The results showed that miR-145 was downregulated in ESCC tumor tissues and cells, while REV3L was upregulated in ESCC tumor tissues. Overexpression of miR-145 decreased REV3L mRNA and protein level in ESCC cell line KYSE150, while decreased miR-145 increased REV3L mRNA and protein level in esophageal epithelium cell line (HEEC). In addition, the luciferase activity of ESCC cells was decreased after the treatment of miR-145 mimic and mRNA 3'UTR-WT. Overexpressed miR-145 significantly inhibited cell viability and elevated cell apoptosis rate upon 5-FU treatment. Additionally, transfection of miR-145 mimic further altered expression of key genes involved in cell apoptosis (Bcl-2, Bax, Caspase3) in ESCC cells treated with 5-FU. miR-145 might be a therapeutic target for the treatment of ESCC.