The high-mobility group nucleosome-binding domain 5 (HMGN5) is a member of the high-mobility group proteins family. Previous study found that HMGN5 is required for tumorigenesis in vitro, and aberrations in the expression of HMGN5 were found in human osteosarcoma, prostate cancer, and squamous cell carcinoma. Nevertheless, the role of HMGN5 in breast cancer remains unclear. This study aimed to investigate the expression and clinical significance of HMGN5 in human breast cancer, confirm the oncogenic role of HMGN5, and explore the mechanism by which HMGN5 contributes to invasion and metastasis. HMGN5 expression was detected in breast cancer tissues and corresponding adjacent non-cancerous tissues from 43 patients by immunohistochemistry, and the clinicopathologic characteristics of all patients were also analyzed. Next, knockdown of HMGN5 protein in MDA-MB-231 cells was performed through a small interfering RNA (siRNA) technique, and cell viability, apoptosis, and invasion were detected by cell vitality test, flow cytometry, and transwell assay, respectively. Immunohistostaining showed that HMGN5 were highly expressed in the nucleus in all breast cancer tissues as compared with the adjacent non-cancerous tissues (ANCT;(73.5 ± 11 vs. 31.0 ± 5 %, P < 0.01). HMGN5 expression level was associated with the poorly differentiated tumor cells, lymph node involvement tumor, and T4 staging tumor. Knockdown of HMGN5 inhibited cell growth, suppressed invasion, and increased cell apoptosis in human breast cancer MDA-MB-231 cells. Western blot analysis demonstrated that the expressions of PCNA, connective tissue growth factor (CTGF), and MMP-9 were decreased in human breast MDA-MB-231 cells depleted of HMGN5. In addition, the apoptotic markers (cleaved PARP and cleaved caspase-3) were significantly increased by HMGN5 knockdown, but microtubule-associated protein 1 light chain 3-II/I (LC3-II/I) did not alter. HMGN5 plays an oncogenic role in human breast cancer by inhibiting cell proliferation and invasion, and activating apoptosis, which could be exploited as a target for therapy in human breast cancer.