We previously demonstrated that cells isolated from the mesenchymal region of the human amniotic membrane (human amniotic mesenchymal tissue cells, hAMTC) possess immunoregulatory roles, such as inhibition of lymphocyte proliferation and cytokine production, and suppression of generation and maturation of monocyte-derived dendritic cells, as reported for MSC from other sources. The precise factors and mechanisms responsible for the immunoregulatory roles of hAMTC remain unknown. In this study, we aimed to identify the soluble factors released by hAMTC and responsible for the anti-proliferative effect on lymphocytes, and the mechanisms underlying their actions, in vitro. Conditioned medium (CM) was prepared under routine culture conditions from hAMTC (CM-hAMTC) and also from fragments of the whole human amniotic membrane (CM-hAM). We analyzed the thermostability, chemical nature, and the molecular weight of the factors likely responsible for the anti-proliferative effects. We also evaluated the participation of cytokines known to be involved in the immunomodulatory actions of MSC from other sources, and attempted to block different synthetic pathways. We demonstrate that the inhibitory factors are temperature-stable, have a small molecular weight, and are likely of a non-proteinaceous nature. Only inhibition of cyclooxygenase pathway partially reverted the anti-proliferative effect, suggesting prostaglandins as key effector molecules. Factors previously documented to take part in the inhibitory effects of MSCs from other sources (HGF, TGF-β, NO and IDO) were not involved. Furthermore, we prove for the first time that the anti-proliferative effect is intrinsic to the amniotic membrane and cells derived thereof, since it is manifested in the absence of stimulating culture conditions, as opposed to MSC derived from the bone marrow, which possess an anti-proliferative ability only when cultured in the presence of activating stimuli. Finally, we show that the amniotic membrane could be an interesting source of soluble factors, without referring to extensive cell preparation.