Acquired chemoresistance of tumor cells is an unwanted consequence of cancer treatment. Overcoming chemoresistance is particularly important for efficiently improving cancer therapies. Here, using multiple lines of evidence, we report the suppressive role of SERTAD1 in apoptosis/anoikis. Among various breast cancer cell lines, higher SERTAD1 expression was found in MCF7 and MDA-MB-231 in suspension than in adherent cell culture. We revealed an unexpected phenomenon that different types of cell deaths were induced in response to different doses of doxorubicin (Dox) in breast cancer cells, presumably via lysosomal membrane permeabilization. A low dose of Dox highly activated autophagy, while a high dose of the chemotherapy induced apoptosis. Inhibition of SERTAD1 promoted the sensitivity of breast cancer cells to Dox and paclitaxel, leading to a significant reduction in tumor volumes of xenograft mice. Simultaneously targeting cancer cells with Dox and autophagy inhibition successfully induced higher apoptosis/anoikis. The novel role of SERTAD1 in maintaining cellular homeostasis has also been suggested in which lysosomal contents, including LAMP1, LAMP2, CTSB, and CTSD, were reduced in SERTAD1-deficient cells.