Purpose: Mesenchymal stromal cells are multipotent progenitor cells residing in connective tissues with the capacity to differentiate into cartilage, bone, muscle or adipose tissue. Besides their differentiation potential, MCSs can also regulate immunoresponses in a paracrine manner by both cell-cell contact mechanisms and the release of soluble immunomodulators. In fact, during the last years, the anti-inflammatory and immunoregulatory properties of MSCs have gained more attention than their differentiation properties. Despite the increased awareness about the potential of MSCs to promote a tissue-healing microenvironment, the molecular mechanisms are not completely understood, and the predictable divergent immunoregulatory potential of MSCs from different sources has not been addressed. In this study, we compare the immunoregulatory properties of culture supernatants from MSCs isolated from different tissues and do large-scale profiling of the secretomes to characterize the spectrum of released soluble proteins from each cell type. Methods: A mixed enzymatic-explant method was used to isolate cells from different tissues including cartilage (ACs), Hoffa's fat pad (HFPCSs), synovial membrane (SMCSs) and umbilical cords (UCSCs). Stromal cells were characterized for MSCs surface markers according to international society for cellular therapy guidelines. To study the cell secretomes, supernatants from monolayer cultures of all cell types at third passage were collected and studied by large-scale quantitative analyses using quantitative label-free LC/MS-MS spectrometry. We also investigated groups of selected cytokines, chemokines, growth factors and proteases involved in tissue remodelling and inflammation in the cell supernatants by multiplex antibody-based immunodetection. Furthermore, in vitro functional assays using mitogen-activated lymphocytes were applied to investigate the immunomodulatory potential of cell supernatants. Results: All stromal cells expressed classical MSCs surface markers. Secretome analyses revealed more proteins identified in the supernatants of ACs (709) compared to HFPSCs (641), SMSCs (567) and UCSCs (653). Hierarchical clustering of identified proteins showed two major clusters with UCSCs in one cluster and stromal cells from adult joint tissues in another cluster. Qualitative comparison of identified proteins using gene ontology biological process databases showed similar numbers of proteins involved in the catabolic process (25% in ACs and 27% in all other stromal cells) and immunoregulation (19% in both ACs and UCSCs, 20% in HFPSCs and 21% in SMSCs). However, comparative expression of selected proteins involved in ECM remodelling revealed the presence of MMP1 in UCSCs at a significantly low level compared to stromal cells from adult joint tissues. In addition, other identified MMPs including MMP2, MMP3, MMP7, MMP10 and MMP14 were not detected in the cell supernatants of UCSCs. The presence of TGF-β1 was also higher in the UCSCs compared to other stromal cells. In line with these observations, quantitative determination of selected cytokines and MMPs using Luminex and Elisa also revealed the significantly lower concentration of IL-17 and MMPs including MMP1, MMP3, MMP7 and MMP9 in the culture supernatants of UCSCs. Importantly, the concentration of TGF-β1 and PGE-2 was significantly higher in the UCSCs. Mitogen-activated lymphocyte functional assay also confirmed significant blocking of lymphocyte proliferation and migration in the presence of conditioned medium (CM) from UCSCs compared to CM from ACs, HFPSCs and SMSCs. Furthermore, lymphocytes secretion of TNF-α and IFN-γ was significantly decreased in the presence of UCSCs-CM. Conclusions: Our results show that the secretome of culture expanded UCSCs is considerably different than the secretome of stromal cells harvested from adult tissues, demonstrating enhanced expression of immunosuppressive molecules and reduced expression of proteases and catabolic factors. In functional lymphocyte activation assays, only conditioned medium from UCSCs exerted suppressive effects. Collectively, our data suggest that UCSCs have superior immunoregulatory properties and would represent the best cell source to promote a tissue-healing microenvironment.