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Prostate cancer (PCa) is the second most prevalent cancer in men worldwide. Most cases of death from PCa are due to metastasis. Early stages of metastasis are mediated by epithelial-mesenchymal transition (EMT) process through which cancer cells acquire motility and invasive characteristics. Thus, more potent and novel therapeutic strategies must be designed based on the inhibition of EMT or metastasis. Herein, we employ a co-culture system to evaluate the anti-EMT effects of human amniotic mesenchymal stromal cells (hAMSCs) on LNCaP PCa cells. The RNA of treated (sample) and untreated cancer cells (control) and whole-cell lysates of related cells were prepared and analysed through quantitative real-time polymerase chain reaction (qRT-PCR) and western blot, respectively. Based on the results, the expression of vimentin, Snail and Zeb1 in LNCaP cells decreased and the expression of E-cadherin increased after treatment with hAMSCs. Furthermore, induction of the cellular apoptosis in LNCaP cells was detected. The anti-cancer activity of conditioned medium from hAMSCs was shown using hanging drop technique (a 3D cell culture model). Our findings support the idea that stem cells can be considered as a novel therapeutic approach to inhibit prostate cancer cells. SIGNIFICANCE OF THE STUDY: The anti-tumour activity of hAMSCs on LNCaP prostate cancer cells using 2D and 3D cell culture models via induction of apoptosis, suppression of EMT process and down-regulation of EGFR was shown. The results of the present study support this idea that hAMSCs may be a potent therapeutic tool to suppress tumour growth in LNCaP prostate cancer cells., (© 2021 John Wiley & Sons Ltd.)