SAHA, an inhibitor of histone deacetylase activity, has been shown to sensitize tumor cells to apoptosis induced by TRAIL, a member of TNF-family. In this paper we investigated the effect of SAHA/TRAIL combination in two breast cancer cell lines, the ERα-positive MCF-7 and the ERα-negative MDA-MB231. Treatment of MDA-MB231 and MCF-7 cells with SAHA in combination with TRAIL caused detachment of cells followed by anoikis, a form of apoptosis which occurs after cell detachment, while treatment with SAHA or TRAIL alone did not produce these effects. The effects were more evident in MDA-MB231 cells, which were chosen for ascertaining the mechanism of SAHA/TRAIL action. Our results show that SAHA decreased the level of c-FLIP, thus favouring the interaction of TRAIL with the specific death receptors DR4 and DR5 and the consequent activation of caspase-8. These effects increased when the cells were treated with SAHA/TRAIL combination. Because z-IEDT-fmk, an inhibitor of caspase-8, prevented both the cleavage of the focal adhesion-kinase FAK and cell detachment, we suggest that activation of caspase-8 can be responsible for both the decrement of FAK and the consequent cell detachment. In addition, treatment with SAHA/TRAIL combination caused dissipation of ΔΨ(m), activation of caspase-3 and decrement of both phospho-EGFR and phospho-ERK1/2, a kinase which is involved in the phosphorylation of BimEL. Therefore, co-treatment also induced decrement of phospho-BimEL and a concomitant increase in the dephosphorylated form of BimEL, which plays an important role in the induction of anoikis. Our findings suggest the potential application of SAHA in combination with TRAIL in clinical trials for breast cancer.