Alternative splicing of estrogen receptor β (ERβ) yields five isoforms, but their functions remain elusive. ERβ isoform 5 (ERβ5) has been positively correlated with better prognosis and longer survival of patients with breast cancer (BCa) in various clinical studies. In this study, we investigated the inhibitory role of ERβ5 in BCa cells. Although ERβ5 does not reduce proliferation of BCa cell lines MCF-7 and MDA-MB-231, its ectopic expression significantly decreases their survival by sensitizing them to doxorubicin- or cisplatin-induced apoptosis through the intrinsic apoptotic pathway. Moreover, we discovered Bcl2L12, which belongs to the Bcl-2 family regulating apoptosis, to be a specific interacting partner of ERβ5, but not ERβ1 or ERα, in an estradiol-independent manner. Knockdown of Bcl2L12 enhanced doxorubicin- or cisplatin-induced apoptosis, and this process was further promoted by ectopic expression of ERβ5. Whereas Bcl2L12 was previously shown to inhibit apoptosis through binding to caspase 7, such interaction is reduced in the presence of ERβ5, suggesting a mechanism by which ERβ5 sensitizes cells to apoptosis. In conclusion, ERβ5 interacts with Bcl2L12 and functions in a novel estrogen-independent molecular pathway that promotes chemotherapeutic Agent-Induced in vitro apoptosis of BCa cell lines.