BACKGROUND: Breast cancer remains the most invasive female malignancy worldwide. Functional role of microRNA-940 (miR-940) have been investigated in various cancer. The purpose of this study was to assess the serum miR-940 expression and its clinical significance in breast cancer. METHODS: Expression of miR-940 was measured by quantitative real-time polymerase chain reaction (qRT-PCR). The diagnostic value of miR-940 was analyzed with receiver operating characteristics (ROC) analysis. To explore the prognostic performance of miR-940, Kaplan-Meier survival assay and Cox regression analysis were performed. RESULTS: Downregulated miR-940 was detected in the breast cancer patients compared with the healthy controls (P< 0.001). The miR-940 expression was correlated with lymph node metastasis (P= 0.014) and TNM stage (P= 0.003). The area under the ROC curve (AUC) was 0.905, with sensitivity and specificity of 94.5% and 78.6%. From the survival curves, patients with low miR-940 expression had poor overall survival compare with those with high expression (log-rank P= 0.009). The Cox analysis indicated that miR-940 was an independent prognostic factor (HR = 2.645, 95% CI = 1.426–4.906 and P= 0.002). Decreased miR-940 expression was also been found in triple-negative breast cancer (TNBC) samples, and might predict poor prognosis in TNBC patients. CONCLUSIONS: Serum downregulated miR-940 may serve as a reliable diagnostic and prognostic biomarker in breast cancer patients.