Osteogenic differentiation of human mesenchymal stem cells (hMSCs) is regulated by multiple transcription factors and signaling molecules. However, the molecular mechanisms underlying this process remain to be fully elucidated. MicroRNAs (miRNAs) act as key regulators in various biological processes by mediating mRNA degradation or translational inhibition of target genes. In this study, we report that miR-346 plays critical roles in regulating osteogenic differentiation of hBMSCs. The expression of endogenous miR-346 was increased during osteogenic differentiation of hBMSCs. Overexpression of miR-346 significantly promoted osteogenic differentiation, whereas miR-346 depletion suppressed this process. Further studies confirmed that miR-346 directly targeted the 3'-UTR of the glycogen synthase kinase-3β (GSK-3β) gene so as to suppress the expression of GSK-3β protein. Similar to miR-346 overexpression, GSK-3β depletion promoted osteogenic differentiation, whereas GSK-3β overexpression reversed the promotional effect of miR-346. We further found that miR-346 overexpression activated the Wnt/β-catenin pathway and increased the expression of several downstream genes including CyclinD1, c-Myc, TCF-1 and LEF-1. Depletion of β-catenin almost completely blocked the positive role of miR-346 on osteogenic differentiation. Taken together, our data indicate that miR-346 positively regulates hBMSC osteogenic differentiation by targeting GSK-3β and activating the Wnt/β-catenin pathway.