In the tumor microenvironment, soluble molecules play important role in the establishment of a pre-metastatic niche. The S100 calcium-binding protein family are inflammatory molecules that contribute to the development of a pro-inflammatory tumor microenvironment. S100B belongs to the S100 family and serum S100B (also known as S100beta) serves as a marker for metastasis in lung cancer, ovarian cancer and melanoma. However, the association between S100B and the metastasis of breast cancer is not yet well understood. In the present study, a relatively low S100B expression was observed in the tumor samples compared to normal breast tissue among online microarray datasets. When the estrogen receptor (ER)-negative breast cancer cell lines, MDA-MB-231 and Hs578T, were treated with recombinant human S100B, cell migration was significantly inhibited and epithelial cadherin expression was increased. Our results revealed that a high S100B expression predicted a good overall survival in patients with ER-negative breast cancer, and good distant metastases-free survival in all patients with breast cancer via the analysis of the KM plotter and SurvExpress databases. Although previous studies have indicated that the interaction of S100B with wild-type p53 inhibits p53 function, a high S100B expression is associated with a good prognosis in patients with p53 mutant and p53 wild-type breast cancers. On the whole, our findings demonstrate that S100B treatment suppresses the migratory capacity of ER-negative breast cancer and that S100B expression may serve a predictive marker for metastasis in breast cancer.