Human amniotic epithelial cells (hAECs) have been the object of intense research due to their potential therapeutic use. In this paper, we present molecular evidence of a bona fide epithelial to mesenchymal transition (EMT) undergone by hAECs. Amniotic membrane (AM)-derived hAECs showed the presence of typical epithelial markers such as E-cadherin and cytokeratins. hAECs in culture, however, underwent morphological changes acquiring a mesenchymal shape. Epithelial cell markers were lost and typical mesenchymal markers, such as vimentin and α-SMA, appeared. Several genes associated with EMT, such as SNAI1, MMP9, PAI1, or ACTA2, increased their expression. The expression of the transcription activators KLF4 or MTA3 was consistent with the downregulation of CDH1. We have shown that hAECs undergo EMT due to the autocrine production of TGF-β. Furthermore, the addition of the TGF-β receptor I (ALK5) inhibitor SB-431542 or TGF-β neutralizing antibody to hAECs prevented EMT and preserved the hAECs' epithelial phenotype. Altogether, these results suggest that cultured hAECs undergo EMT through the autocrine production of TGF-β.