The present study aimed to investigate the efficacy of five signaling pathway inhibitors, N-[N-(3,5‑difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester, vismodegib, salinomycin, ruxolitinib and stattic, as novel therapeutic agents that target breast cancer stem cells (BCSCs) in triple-negative breast cancer (TNBC). The in vitro anti-proliferative, anti-invasive, pro-apoptotic and inhibitory effects on BCSC self-renewal of these signaling pathway inhibitors on the TNBC stem cell line HCC38 were examined by MTT assays, Matrigel invasion assays, flow cytometry and suspension mammosphere assays, respectively. For the in vivo study, another TNBC stem cell line, HCC1806, pretreated with these signaling pathway inhibitors, was inoculated into female nonobese diabetic/severe combined immunodeficient mice, and the tumor volumes were measured following tumor formation. Treatment of HCC38 cells with each signaling pathway inhibitor significantly decreased TNBC cell proliferation, cell invasion and mammosphere formation while inducing cell apoptosis by inhibiting the protein expression or phosphorylation of downstream signaling molecules. In the xenograft mouse models, tumor formation and growth of HCC1806 cells pretreated with each signaling pathway inhibitor were effectively suppressed. Treatment with these signaling pathway inhibitors may provide a novel therapeutic strategy against TNBC by targeting BCSCs, thus providing promising insight for clinical applications in patients with TNBC.