AbstractLittle knowledge exists about the mechanisms by which estrogen can impede chemotherapy-induced cell death of breast cancer cells. 17β-Estradiol (E2) hinders cytotoxic drug–induced cell death in estrogen receptor–positive (ER+) breast cancer cells. We noted that the activity of the proapoptotic mixed lineage kinase 3 (MLK3) kinase was relatively higher in estrogen receptor–negative (ER−) breast tumors, suggesting that E2 might inhibit MLK3 activity. The kinase activities of MLK3 and its downstream target, c-Jun NH2-terminal kinase, were rapidly inhibited by E2 in ER+ but not in ER− cells. Specific knockdown of AKT1/2 prevented MLK3 inhibition by E2, indicating that AKT mediated this event. Furthermore, MLK3 inhibition by E2 involved phosphorylation of MLK3 Ser674 by AKT, attenuating the proapoptotic function of MLK3. We found that a pan-MLK inhibitor (CEP-11004) limited Taxol-induced cell death and that E2 accentuated this limitation. Taken together, our findings indicate that E2 inhibits the proapoptotic function of MLK3 as a mechanism to limit cytotoxic drug–induced death of ER+ breast cancer cells. Cancer Res; 70(4); 1731–40