Chemoresistance remains a major obstacle to successful treatment of breast cancer. Although soluble tumor necrosis factor-alpha (sTNF-alpha) has been implicated in mediating drug-resistance in human cancers, whether transmembrane tumor necrosis factor-alpha (tmTNF-alpha) plays a role in chemoresistance remains unclear. Here we found that over 50% of studied patients expressed tmTNF-alpha at high levels in breast cancer tissues and tmTNF-alpha expression positively correlated with resistance to anthracycline chemotherapy. Alteration of tmTNF-alpha expression changed the sensitivity of primary human breast cancer cells and breast cancer cell lines to doxorubicin (DOX). Overexpression of N-terminal fragment (NTF) of tmTNF-alpha, a mutant form with intact intracellular domain of tmTNF-alpha to transmit reverse signals, induced DOX-resistance. Mechanistically, the tmTNF-alpha/NTF-ERK-GST-p axis and tmTNF-alpha/NTF-NF-kappa B-mediated anti-apoptotic functions were required for tmTNF-alpha-induced DOX-resistance. In a xenograft mouse model, the combination of tmTNF-alpha suppression with chemotherapy significantly enhanced the efficacy of DOX. Our data indicate that tmTNF-alpha mediates DOX-resistance through reverse signaling and targeting tmTNF-a may be beneficial for the treatment of DOX-resistant breast cancer.