Metastasis to bone is a frequent problem of advanced breast cancer. Particularly breast cancers, which do not express estrogen receptor α (ERα) and progesterone receptor (PR) and which have no overexpression of human epidermal growth factor receptor 2 (HER2), so-called triple-negative breast cancers (TNBCs), are considered as very aggressive and have a poor prognosis. Recently we have shown that breast cancer cell invasion was dramatically increased when co-cultured with MG63 osteoblast-like cells. Using this model we have now analyzed whether estrogen receptor β (ERβ) plays a role in TNBC cell invasion in vitro. ERα and ERβ protein expression was analyzed using western blot analysis. Invasion was quantified by assessment of TNBC cell migration rate through an artificial basement membrane in a modified Boyden chamber during co-culture with MG63 osteoblast-like cells. The effects of ERβ agonist treatment on CXC motif chemokine receptor 4 (CXCR4) protein expression during co-culture with MG64 cells was quantified using western blot analysis. Proliferation was measured using alamarBlue assay. TNBC cell lines HCC1806 and HCC1937 showed no ERα but high ERβ protein expression. Cell invasion of HCC1806 and HCC1937 TNBC cells was significantly increased when co-cultured with MG63 osteoblast-like cells. Treatment with ERβ selective estrogen agonists liquiritigenin and ERB-041 reduced the ability to invade a reconstituted basement membrane and to migrate in response to the cellular stimulus. During co-culture CXCR4 protein expression of TNBC cell lines HCC1806 and HCC1937 was significantly increased. Treatment with liquiritigenin resulted in a significant decrease of CXCR4 protein expression. Both ERβ agonists showed no effect on TNBC cell proliferation. Our findings suggest that ERβ plays a major role in TNBC invasion. Bone-directed invasion can be inhibited by ERβ agonists.