The type III transforming growth factor-β (TGF-β) receptor (TβRIII), a coreceptor of the TGF-β superfamily, is known to bind TGF-βs and regulate TGF-β signaling. However, the regulatory roles of TβRIII in TGF-β-induced mesenchymal stem cell (MSC) chondrogenesis have not been explored. The present study examined the effect of TβRIII RNA interference (RNAi) on TGF-β3-induced human MSC (hMSC) chondrogenesis and possible signal mechanisms. A lentiviral expression vector containing TβRIII small interfering RNA (siRNA) (SiTβRIII) or a control siRNA (SiNC) gene was constructed and infected into hMSCs. The cells were cultured in chondrogenic medium containing TGF-β3 or control medium. TβRIII RNAi significantly enhanced TGF-β3-induced chondrogenic differentiation of hMSCs, the ratio of type II (TβRII) to type I (TβRI) TGF-β receptors, and phosphorylation levels of Smad2/3 as compared with cells infected with SiNC. An inhibitor of the TGF-β signal, SB431542, not only inhibited TβRIII RNAi-stimulated TGF-β3-mediated Smad2/3 phosphorylation but also inhibited the effects of TβRIII RNAi on TGF-β3-induced chondrogenic differentiation. These results demonstrate that TβRIII RNAi enhances TGF-β3-induced chondrogenic differentiation in hMSCs by activating TGF-β/Smad2/3 signaling. The finding points to the possibility of modifying MSCs by TβRIII knockdown as a potent future strategy for cell-based cartilage tissue engineering.