Prostate cancer is considered a heterogeneous disease depending on clinical, morphological, and molecular perspectives. Prostate cancer relies on androgen and androgen receptors for its survival and growth; therefore, androgen deprivation therapy combined with other approaches is used to treat prostate cancer. Nonetheless, 20–30% of prostate cancer patients evolve an androgen deprivation therapy resistance phenotype, which is a major challenge for physicians. Autophagy is an evolutionary conserved catabolic mechanism that has a double-edged sword role in cancer. At the initial stages of cancer development, autophagy functions as a tumor suppressor via the degradation of potentially oncogenic molecules. Nevertheless, at the advanced stages, autophagy promotes tumor cell survival, metabolic reprogramming, metastasis, and drug resistance due to its interconnection with other distinct cellular and metabolic pathways. This study investigated the cytotoxic, antiproliferative, anti-survival, and anti-migratory effect of autophagy inhibitor chloroquine on metastatic androgen-independent PC-3 prostate cancer cells. A dose-dependent MTT assay was applied to evaluate the cytotoxic and antiproliferative effect of chloroquine on human PC-3 prostate cancer cells following 48 hours of treatment. The IC50 value was determined as 53.281 μM for chloroquine. Treatment of PC-3 cells with chloroquine led to significant downregulation of cell proliferation and survival as indicated by the significant decrease of colony formation ratio. The effect of chloroquine on PC-3 cell migration was validated using a cell motility assay and the findings indicated a significant suppression of PC-3 cell migration. These results revealed a significant antiproliferative, anti-survival, and anti-migratory effect of chloroquine on PC-3 prostate cancer cells.