This study was performed to investigate osteoblastogenesis of human mesenchymal stem cells (hMSCs) cultured in 3-D scaffolds stimulated with low-intensity pulsed ultrasound and to identify the underlying mechanism mediated by soluble receptor activator of nuclear factor kappa B ligand (sRANKL) secreted by hMSCs. The results indicate that the mRNA levels of core-binding factor subunit alpha subunit 1 (CBFA1), osterix (OSX), alkaline phosphatase (ALP), osteocalcin and osteoprotegerin (OPG) and sRANKL production of hMSCs stimulated by ultrasound were significantly increased compared with the levels without ultrasound stimulation. Attenuating the sRANKL activity of ultrasound-treated hMSCs significantly reduced the mRNA expression of CBFA1, OSX, ALP and OPG. Adding sRANKL in hMSC culture significantly increased the mRNA expression of CBFA1, OSX and OPG. Together, the results suggest that osteoblastogenesis of hMSCs enhanced by ultrasound stimulation is mediated by endogenous sRANKL.