Toll-like receptor 3 (TLR3) is an endosomal receptor expressed in several immune and epithelial cells. Recent studies have highlighted its expression also in solid tumours, including prostate cancer (PCa), and described its role mainly in the pro-inflammatory response and induction of apoptosis. It has been found upregulated in some castration-resistant prostate cancers. However, the role of TLR3 in prostate cancer progression remains largely unknown. We have experimentally demonstrated that exogenous TLR3 activation in PCa cell lines leads to the significant induction of secretion of the cytokines IL-6, IL-8 and IFN-β depending on the model and chemoresistance status. Transcriptomic analysis of TLR3 overexpressing cells revealed a functional program that is enriched for genes involved in the regulation of cell motility, migration, and tumour invasiveness. Increased motility, migration, and invasion in TLR3 overexpressing cell line were confirmed by several in vitroassays and using an orthotopic prostate xenograft model in vivo. Furthermore, TLR3-ligand induced apoptosis via cleavage of caspase-3/7 and PARP, predominantly in TLR3 overexpressing cells. We conclude that TLR3 may be involved in prostate cancer progression and metastasis, however, it might also represent an Achilles' heel of PCa which can be exploited for targeted therapy.