Breast cancer is considered the most frequent cancer among females worldwide. Breast cancer is a heterogeneous disease that can be curable in its early stages. Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is characterized by the absence of progesterone receptor (PR), estrogen receptor (ER), and human epidermal growth factor receptor 2 (HER2). Autophagy is an intracellular macromolecular degradation mechanism that is involved in the recycling of macromolecules, providing energy sources for normal and cancer cells. In cancer cells, autophagy acts as a tumor suppressor in the early stages of cancer development by degrading potentially oncogenic substances. However, autophagy is associated with prolonged survival and tumorigenesis in the late stages of cancer development due to its linkage with various cellular and metabolic processes. This study examined the cytotoxic, antiproliferative, anti-survival and anti-migratory effect of autophagy inhibitor chloroquine on human metastatic MDA-MB-231 breast cancer cells. The antiproliferative effect of chloroquine on human metastatic MDA-MB-231 breast cancer cells was determined by dose-dependent MTT assay after 48 hours of treatment. The IC50 value of chloroquine was defined to be 26.832 μM Treatment of MDA-MB-231 cells with chloroquine led to significant inhibition of cellular proliferation and survival as estimated by the remarkable reduction of colony formation ratio. Cell motility assay was used to estimate the effect of chloroquine on MDA-MB-231 cell migration and the findings exhibit a significant suppression of migration. These results validated a marked antiproliferative, anti-survival and anti-migratory effect of chloroquine on human metastatic MDA-MB-231 breast cancer cells.