Abnormal activation of Nuclear factor kappa B (NF-κB) signaling pathway is closely implicated in triple-negative breast cancer (TNBC) growth, metastasis and tumor immune escape. In this study, the anti-cancer effects of icariin, a natural flavonol glycoside, towards breast cancer cells and the underlying mechanisms were investigated. This investigation showed that icariin selectively inhibited proliferation and triggered apoptosis in breast cancer cells in a concentration- and time-dependent manner, but exhibited little cytotoxicity in normal breast cells. Moreover, icariin induced cell apoptosis via a mitochondria-mediated pathway, as indicated by upregulated ratio of Bax/Bcl-2 and ROS induction. Importantly, icariin impaired activation of NF-κB/EMT pathway, as evidenced by upregulation of SIRT6, resulting in inhibition of migration and invasion of breast cancer cells. Additionally, oss-128167, an inhibitor of SIRT6, dramatically attenuated anti-migration and anti-invasion effects of icariin. Transcriptomic analysis verified that impairment of NF-κB leads to the selective function of icariin in breast cancer cells. Notably, icariin exhibited a significant tumor growth inhibition and anti-pulmonary metastasis effect in a tumor mouse model of MDA-MB-231 and 4T1 cells by regulating tumor immunosuppressive microenvironment. Together, these results showed that icariin could effectively trigger apoptosis and inhibit migration of breast cancer cells via the SIRT6/NF-κB signaling pathway, suggesting that icariin might serve as a potential candidate drug for the treatment of breast cancer. This article is protected by copyright. All rights reserved.