Osteoporosis and osteoarthritis are orthopedic disorders that affect millions of elderly people worldwide; stimulation of bone formation is a potential therapeutic strategy for the treatment of these conditions. As the only bone-forming cells, osteoblasts play a key role in bone reconstruction. The microRNA miR-17-3p is downregulated during osteogenic differentiation of human bone marrow mesenchymal stem cells, but its precise role in this process is unknown. Here, we investigated the role of miR-17-3p in osteoblast differentiation. An in vitro model of osteogenesis was established by treating MC3T3-E1 murine pre-osteoblast cells with bone morphogenetic protein 2 (BMP2). The expression of miR-17-3p in BMP2-induced MC3T3-E1 cells was detected by reverse transcription-quantitative PCR, and its effects on cells transfected with miR-17-3p mimic or inhibitor were evaluated by Alizarin Red staining, alkaline phosphatase (ALP) activity assay, and by detection of osteoblast markers including the ALP, collagen type I α1 chain, and osteopontin genes. Bioinformatics analysis was carried out to identify putative target genes of miR-17-3p, and the luciferase reporter assay was used for functional validation. Rescue experiments were performed to determine whether SRY-box transcription factor 6 (Sox6) plays a role in the regulation of osteoblast differentiation by miR-17-3p. We report that miR-17-3p was downregulated upon BMP2-induced osteoblast differentiation in MC3T3-E1 cells, and this was accompanied by decreased differentiation and mineralization, ALP activity, and expression of osteogenesis-related genes. Sox6 was confirmed to be a target gene of miR-17-3p in osteoblasts, and the inhibitory effect of miR-17-3p on osteoblast differentiation was observed to occur via Sox6. These results suggest the existence of a novel mechanism underlying miRNA-mediated regulation of osteogenesis, which has potential implications for the treatment of orthopedic disorders. This article is protected by copyright. All rights reserved.