NUDT21, an RNA binding protein, has been reported to play an important role in the regulation of multiple biological responses. Detection of NUDT21 expression may lead to the identification of a novel marker for breast cancer.The aim of this study was to investigate the clinical significance and functional role of NUDT21 in breast cancer.The protein expression of NUDT21 was examined by immunohistochemistry (IHC) in 100 paraffin-embedded, archived breast cancer samples and 100 benign breast tissues. Then, the correlations between the NUDT21 expression and clinicopathologic characteristics and prognoses of the breast cancer patients were analyzed. In addition, the function of NUDT21 in breast cancer cell lines was detected by the methyl thiazolyl tetrazolium, colony formation and transwell assays. Finally, mass spectrometry analysis and Western blotting were used to identify the proteins that interact directly with NUDT21.IHC analysis revealed that the expression of NUDT21 was significantly lower in breast cancer tissues compared with benign breast disease tissues. The correlation analysis revealed that low expression of NUDT21 was positively correlated with tumor size, lymph node metastasis, and TNM stage. Also, Kaplan-Meier survival curves showed that patients with lower NUDT21 expression had shorter overall survival and relapse-free survival compared with higher NUDT21 expression. In addition, the knockdown of NUDT21 enhanced cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT). Consistently, the overexpression of NUDT21 inhibited cell proliferation, migration, invasion, and EMT. In addition, NUDT21 directly interacted with CPSF6 and negatively regulated its expression. Moreover, the knockdown of CPSF6 reversed NUDT21 expression-induced cancer cell migration and invasion.NUDT21 might play a tumor-suppressive role by inhibiting cell proliferation and invasion via the NUDT21/CPSF6 signaling pathway in breast cancer cells.