Abstract Background Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gα_i/o protein coupled receptor (GPCR). Methods We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. Results We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gα_i/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. Conclusions Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.