Extracellular matrix (ECM) tumorigenic alterations resulting in high matrix deposition and stiffening are hallmarks of adenocarcinomas and are collectively defined as desmoplasia. Here, we thoroughly analysed primary prostate cancer tissues obtained from numerous patients undergoing radical prostatectomy to highlight reproducible structural changes in the ECM leading to the loss of the glandular architecture. Starting from patient cells, we established prostate cancer tumoroids (PCTs) and demonstrated they require TGF-beta signalling pathway activity to preserve phenotypical and structural similarities with the tissue of origin. By modulating TGF-beta signalling pathway in PCTs, we unveiled its role in ECM accumulation and remodelling in prostate cancer. We also found that TGF-beta-induced ECM remodelling is responsible for the initiation of prostate cell epithelial-tomesenchymal transition (EMT) and the acquisition of a migratory, invasive phenotype. Our findings highlight the cooperative role of TGF-beta signalling and ECM desmoplasia in prompting prostate cell EMT and promoting tumour progression and dissemination.