Mesenchymal stem cells (MSCs) have been applied to cell-based therapy due to their multiple differentiation ability, the low expression of co-stimulatory molecules and immunosuppressive properties. Despite their immunomodulatory role, the issue of the survival and permanence of MSCs at the site of injury has not yet been fully resolved. Therefore, in order to improve the therapeutic potential of MSCs, it is important to study the mechanisms mediating the relative instability of MSCs in clinical trials. The Toll-like receptors (TLRs) are an important component of innate and adaptive immune responses. In this study, we demonstrate that the activation of two TLRs, namely TLR3 and TLR4, in human umbilical cord-derived MSCs (UCMSCs) induces the expression of inflammatory markers. In addition, as shown by our results, TLR3 upregulated the expression of stem cell markers, while TLR4 downregulated their expression. The upregulation in the expression of the inflammatory markers did not alter the immune status of the UCMSCs or mediate the immune attack of the MSCs by allogeneic immune cells. We found that the activation of TLR3 inhibited the differentiation of UCMSCs into osteocytes, while that of TLR4 increased this differentiation to a certain extent. Taken together, the results of this study provide a new role for TLR3 and TLR4 as regulators of the biological functions of UCMSCs.