Human breast tumors comprise a minor sub-population of tumor-initiating cells (TICs), commonly termed cancer stem cells. TICs are thought to sustain tumor growth and to confer resistance to current anticancer therapies. Hence, targeting TIC may be essential to achieving durable cancer cures. To identify molecular targets in breast TIC, we employed a transgenic mouse model of ERBB2 breast cancer; tumors arising in this model comprise a very high frequency of TIC, which is maintained in tumor cell populations propagated in vitro as non-adherent tumorspheres. The Notch pathway is dysregulated in human breast tumors and overexpression of constitutively active Notch proteins induces mammary tumors in mice. The Notch pathway has also been implicated in stem cell processes including those of mammary epithelial stem cells. Hence, we investigated the potential that the Notch pathway is required for TIC activity. We found that an antagonist of Notch signaling, a gamma (γ)-secretase inhibitor termed MRK-003, inhibited the survival of tumorsphere-derived cells in vitro and eliminated TIC as assessed by cell transplantation into syngeneic mice. Whereas MRK-003 also inhibited the self-renewal and/or proliferation of mammosphere-resident cells, this effect of the inhibitor was reversible thus suggesting that it did not compromise the survival of these cells. MRK-003 administration to tumor-bearing mice eliminated tumor-resident TIC and resulted in rapid and durable tumor regression. MRK-003 inhibited the proliferation of tumor cells, and induced their apoptosis and differentiation. These findings suggest that MRK-003 targets breast TIC and illustrate that eradicating these cells in breast tumors ensures long-term, recurrence-free survival.