Human DNA polymerase β (polβ) is a small, monomeric protein essential for short‐patch base excision repair (BER). polβ plays an important role in the regulation of chemotherapy sensitivity in tumour cells. In this study, we determined that the expression levels of polβ mRNA and miR‐149 in tumour tissues were significantly higher than in adjacent non‐tumour tissues. We also found that the expression level of miR‐149 in EC tumour tissues was inverse to that of polβ expression. Bioinformatics analysis and dual‐luciferase reporter assay predicted that miR‐149 negatively regulates polβ expression by directly binding to its 3′UTR. CCK‐8 assay indicated that miR‐149 could enhance the anti‐proliferative effects of cisplatin in EC1 and EC9706 cell lines. Flow cytometry, caspase 3/7 activity, and immunofluorescence microscopy results indicated that miR‐149 could enhance the apoptotic effects of cisplatin in EC1 and EC9706 cell lines. We also showed that the expression of polβ lacking the 3′UTR sequence could override the proliferative and apoptotic functions of miR‐149, suggesting that miR‐149 negatively regulates polβ expression by binding to its 3′UTR. Surface plasmon resonance results also showed that miR‐149 could bind with wild‐type polβ. In addition, we identified a new variant of polβ (C1134G). In conclusion, this study confirms that miR‐149 may enhance the sensitivity of EC cell lines to cisplatin by targeting polβ, and that miR‐149 may be unable to regulate the C1134G variant of polβ. Based on these findings, potential drugs could be developed with a focus on enhanced sensitivity of EC patients to chemotherapy.