Osteogenic differentiation of human adipose tissue-derived stem cells (hASCs) is a complex process that is regulated by multiple factors, including microRNAs (miRNAs). The miRNA miR-20a was shown to promote bone formation from bone marrow-derived mesenchymal stem cells. However, the role of miR-20a in osteogenic differentiation of hASCs remains unclear. In this study, we systematically evaluated the function of miR-20a in regulating hASC osteogenesis in vitro. hASCs were transduced with miR-20a-overexpressing and miR-20a-sponge lentiviral vectors, with green fluorescent protein (GFP) as a control. The results showed that miR-20a transcription was upregulated after hASC mineralization. Compared with the miR-20a-sponge, GFP, and hASC groups, the miR-20a-overexpressing group showed higher alkaline phosphatase (ALP) activity on days 7 and 14. Moreover, the mRNA level of ALP increased significantly in the miR-20a-overexpressing group on day 14. Furthermore, the protein of the target gene PPAR gamma was decreased, and the osteogenic differentiation-associated proteins ALP, osteocalcin, and RUNX2 were upregulated. hASCs anchored to HA/beta-TCP revealed a healthy polygonal morphology and developed cytoplasmic extensions. miR-20a promoted osteogenic differentiation of the cell scaffold. Taken together, these data -confirm that miRNA-20a promotes the osteogenesis of hASCs in vitro, and its essential role in vivo needs further -investigation.