There is a need for the development of new safe and efficacious drug therapies for the treatment of estrogen receptor (ER)‑negative breast cancers. 1-Methyl-3,5-bis[(E)-4-pyridyl)methylidene]-4-piperidone (RL66) is a second generation curcumin analog that exhibits potent cytotoxicity towards a variety of ER-negative breast cancer cells. Therefore, we have further examined the mechanism of this novel drug in in vitro and in vivo models of ER-negative breast cancer. The mechanistic studies demonstrated that RL66 (2 µM) induced cell cycle arrest in the G2/M phase of the cell cycle. Moreover, RL66 (2 µM) caused 40% of SKBr3 cells to undergo apoptosis after 48 h and this effect was time-dependent. This correlated with an increase in cleaved caspase-3 as shown by western blot analysis. RL66 (2 µM) also decreased HER2/neu phosphorylation and increased p27 in SKBr3 cells, while in MDA-MB-231 and MDA-MB-468 cells RL66 (2 µM) significantly decreased Akt phosphorylation and transiently increased the stress kinases JNK1/2 and MAPK p38. In addition, RL66 exhibited anti-angiogenic potential in vitro as it inhibited HUVEC cell migration 46% and the ability of these cells to form tube‑like networks. RL66 (8.5 mg/kg) suppressed the growth of MDA-MB-468 xenograft tumors by 48% compared to vehicle control following 10 weeks of daily oral administration. Microvessel density in the tumors from treated mice was also decreased 57% compared to control. Thus our findings demonstrate that RL66 has potent proapoptotic and anti-angiogenic properties in vivo and in vitro and has the potential to be further developed as a drug for the treatment of ER‑negative breast cancer.