Estrogen is a mitogen in most estrogen receptor-α (ERα)-positive breast cancers. We have found that Smad4, a common signal transducer in the transforming growth factor-β superfamily, acts as an ERα transcriptional corepressor. Here, we show that Smad4 induces apoptosis in ERα-positive MCF-7 breast cancer cells, but not in ERα-negative MDA-MB-231 cells. Smad4 induced expression of short Bim isoforms (by alternative splicing) and Bax and release of cytochrome c in ERα-positive cells only, and expression of these apoptotic marker genes was reduced when ERα small interfering RNA was introduced. Notably, Smad4 was able to induce apoptosis in MDA-231 cells with acquired ERα expression. Furthermore, Smad4 inhibited ERα-positive tumor growth by inducing apoptosis in tumor xenografts in nude mice. The sizes of tumors expressing Smad4 were only one-tenth the size of those expressing green fluorescent protein, whereas in ERα-negative cells, Smad4 did not reduce the tumor size. Notably, Smad4 also promoted short Bim isoform and Bax expression and release of cytochrome c only in ERα-positive MCF-7 tumor xenografts. Bim was sufficient for induction of apoptosis, and the short form was the most potent inducer. Our results demonstrate that Smad4 induces apoptosis by regulating Bim splicing as an initial intrinsic signal in ERα-positive cells. Smad4-induced apoptosis in ERα-positive breast cancer cells may explain the invasive nature of ERα-negative breast tumors, thereby providing a potential target for breast cancer intervention. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.