Trefoil factor 1 (TFF1) is a small secretory protein expressed in various types of carcinomas including breast cancer. The TFF1 gene contains an estrogen response element and its expression can be regulated by estrogen. Previous reports showed that TFF1 could protect cells from induced apoptosis in vitro. In the present study, the effect of estrogen on the promotion of doxorubicin-induced apoptosis resistance and the role of TFF1 in this process was demonstrated using the MCF-7 breast cancer cell model. Stable knockdown of the TFF1 gene in MCF-7 cells was generated and used to test the sensitivity to doxorubicin treatment compared to mock control cells in the presence or absence of 17β-estradiol. The apoptotic cells were measured by flow cytometry. The results showed that with the stimulation of apoptosis by doxorubicin, 17β-estradiol could suppress this process in mock cells but not in TFF1 knockdown cells. Moreover, using a viable cell counting method, it was shown that the anti-TFF1 antibody could reverse the anti-apoptotic effect of estrogen in mock cells and recombinant TFF1 could recover doxorubicin-induced cell death in TFF1 knockdown cells. This process, however, could not be inhibited by fulvestrant, an estrogen antagonist. An apoptosis protein array experiment reflected the role of the anti-oxidative enzyme catalase in estrogen and TFF1-modulated apoptosis and this was confirmed by enzymatic assay. These phenomena determine the role of TFF1 in estrogen-promoted resistance to apoptosis induced by doxorubicin in MCF-7 breast cancer cells. The TFF1 gene may be a target for enhancing the sensitivity to chemotherapy in breast cancer treatment.