Understanding the biology of breast cancer stem cells and trying new ways to obliterate these cells would be a key step in developing cures for breast cancer. The objective of this study was to investigate the effect of mutant TNFalpha on human breast cancer stem cells derived from MCF7 cell line under the characterization of biologic features of these cells in vitro. By FACS analysis and sorting, we got MCF7 side population (SP) cells and showed that MCF7 SP cells possess cancer stem cell characteristics using the accepted breast cancer stem cell markers, but do not express multiple drug resistance transporters. Furthermore, by RT-PCR, these stem cells were found to constitutively express TNFR-p55 and TNFR-p75. After being treated with Mt rh471 TNFalpha, SP cells displayed a decreased self-renewal ability and an increased apoptosis by three different methods. When monocolony antibody against TNFR-p55 was added into the culture medium, the inhibitory effect of Mt rh471 TNFalpha on self-renewal was blocked completely, but this was not the case for that of Wt rhTNFalpha. The possible reasons might be that the increased binding of Mt rh471 TNFalpha mainly to TNFR-p55 results in induction of apoptosis of SP cells, while Wt rhTNFalpha could bind to both TNFR-p55 and TNFR-p75 which would lead to NFkappaB activity, resulting in a discounted apoptotic effect. These data suggest that Mt rh471 TNFalpha might be a negative regulator of the breast cancer stem cell-like cells and have the potential to treat breast cancer in clinic.