Objective Previously, we have shown that miRNA-342-3p was increased during osteogenic differentiation of human umbilical mesenchymal stem cells (hUCMSCs) via regulating the sonic hedgehog (Shh) pathway. In this study, our objective is to further investigate the role of miRNA-342-3p in activation of Shh pathway by targeting suppressor of fused protein (Sufu), a suppressor of transcriptional factor Gli, as well as the potential interaction with transforming growth factor beta (TGF-beta) signaling pathway during osteogenic induction of hUCMSCs. Materials and methods HUCMSCs that stable overexpression or knockdown of miRNA-342-3p were established by infection with lentiviral vectors. mRNA and protein levels of Hedgehog signaling pathway and osteogenic genes were measured by RT-qPCR and western blot assays. Luciferase reporter assay was performed to test the direct binding site of Sufu 5 ' UTR targeted by miRNA-342-3p. Results Overexpression of miRNA-342-3p in hUCMSCs enhanced the expression of osteogenic genes by targeting Sufu. And the potential of osteogenic differentiation of hUCMSCs was inhibited while knocking down miRNA-342-3p. Meanwhile, induced the TGF-beta expression level was also observed upon overexpressing miRNA-342-3p, suggesting activation of TGF-beta signaling pathway was a potential mechanism of miRNA-342-3p-mediated osteogenesis in hUCMSCs. Conclusions Our findings provide new mechanistic evidence that miRNA-342-3p might be a valuable therapeutic target in bone regeneration.