Mesenchymal stem cells (MSCs) are now widely used in clinical cell‑based therapy due to their characteristics of low immunogenicity, multiple differentiation potency and the capability to modulate immune responses. However, accumulated research has indicated the absence of engrafted MSCs because of the increased immunogenicity of MSCs. Toll‑like receptors (TLRs) are essential for the innate immune response and regulating the biological function of MSCs. The present study used human umbilical cord‑derived MSCs (UCMSCs) and activated the TLR8 pathway of UCMSCs to study the role of TLR8 in mediating the immune status of UCMSCs. The results demonstrated that the activation of TLR8 increased both the proliferation of peripheral blood mononuclear cells (PBMCs) isolated from healthy human volunteers and the release of lactate dehydrogenase (LDH) in supernatant from the PBMC‑UCMSCs co‑culture system. Reverse transcription-quantitative polymerase chain reaction indicated that the TLR8 agonist increased the expression of many co‑stimulatory molecules and pro‑inflammatory genes, and flow cytometry indicated that activation of the TLR8 agonist increased co‑stimulation protein levels but reduced specific surface markers, as confirmed by the part loss of stemness of UCMSCs. Finally, TLR8 increased osteocyte differentiation but had no effect on chondrocyte and adipocyte differentiation. The current study indicated the implication to TLR8 as regulators of the immunogenicity of UCMSCs.