Dose-dependent toxicity and acquired resistance are two major challenges limiting the efficacious treatment of mammary carcinoma (MC) with doxorubicin. Herein, we investigated the function of Trefoil Factor 3 (TFF3) in the sensitivity and acquired resistance of estrogen receptor positive (ER+) MC cells to doxorubicin. Doxorubicin treatment of ER+MC cells increased TFF3 expression. The depletion of TFF3 by siRNA or inhibition with a small molecule TFF3 inhibitor (AMPC) synergistically enhanced the efficacy of doxorubicin in ER+MC through the suppression of doxorubicin-induced AKT activation and enhancement of doxorubicin-induced apoptosis. Elevated expression of TFF3 and increased activation of AKT were also observed using a model of acquired doxorubicin resistance in ER+MC cells. AMPC partially re-sensitized the doxorubicin resistant cells to doxorubicin-induced apoptosis. Indeed, doxorubicin resistant ER + MC cells exhibited increased sensitivity to AMPC as a single agent compared to doxorubicin sensitive cells. In vivo, AMPC attenuated growth of doxorubicin sensitive ER+MC xenografts whereas it produced regression of xenografts generated by doxorubicin resistant ER+MC cells. Hence, TFF3 inhibition may improve the efficacy and reduce required doses of doxorubicin in ER+MC. Moreover, inhibition of TFF3 may also be an effective therapeutic strategy to eradicate doxorubicin resistant ER+MC.