Metastasis is the leading cause of death for breast cancer patients. Nerve guidance factor 4 (Netrin-4, NTN4) is reduced in variety of malignancies and involved in tumor metastasis. However, the functions of NTN4 and related molecular mechanisms in breast cancer are poorly understood. Oncomine data revealed that NTN4 was decreased in breast cancer compared with normal breast tissues. For fresh frozen breast cancer samples, significantly depressed expression of NTN4 mRNA was observed in lesion tissues compared with that in adjacent tissues. Afterwards, NTN4 protein level was evaluated in 52 paired breast cancer tissues, and the results were consistent with that in fresh frozen samples. NTN4 expression was upregulated using NTN4-pcDNA3.1 plasmid in MDA-MB-231 cells and silenced using NTN4 small interfering RNA (siRNA) in Hs578T cells. Then the effects of NTN4 overexpression or knockdown on breast cancer cell migration and invasion were investigated. The results manifested that NTN4 overexpression attenuated cell migration and invasion, and induced N-cadherin and vimentin downregulation, while NTN4 siRNA-transfected cells had a significant increase in migration and invasion, as well as upregulation in N-cadherin and vimentin expression. These results demonstrate that NTN4 is reduced in breast cancer tissues and NTN4 is associated with breast cancer cell migration and invasion via regulation of epithelial mesenchymal transition (EMT)-related biomarkers.