Bone homeostasis is maintained by balanced osteoblast-mediated tissue production and osteoclastmediated tissue destruction, and is disrupted in pathological conditions such as osteoporosis. The mechanisms underlying osteogenic differentiation of bone marrow mesenchymal stem cells, which is critical to bone homeostasis, are not completely clear, despite extensively studies. Long noncoding RNAs (lncRNAs) have recently emerged as novel therapeutic targets in various diseases. However, the expression pattern and biological function of lncRNAs in osteogenic differentiation remain unclear. In this study, we aimed to determine the role of lncRNAs in osteogenic differentiation of human bone marrow mesenchymal stem cells. We found high lncRNA MCF2L-AS1 expression in human bone marrow mesenchymal stem cells, and used bioinformatics analysis to analyze its function. MCF2L-AS1 knockdown induced inhibition of osteoblast differentiation. Silencing of MCF2L-AS1 increased the expression of miR-33a and subsequently inhibited Runx2 expression at the post-transcriptional level. Moreover, MCF2L-AS1 directly interacted with miR-33a, and downregulation of miR-33a efficiently reversed the suppression of Runx2 induced by MCF2L-AS1 short hairpin RNA (shRNA). Thus, MCF2L-AS1 positively regulated the expression of Runx2 by sponging miR-33a, and promoted osteogenic differentiation in BMSCs. Our results indicated that the lncRNA MCF2L-AS1 plays a critical role in the osteogenic differentiation of BMSCs, and targeting lncRNA MCF2L-AS1 could be a promising strategy to promote osteogenic differentiation.