microRNAs (miRNAs) play pivotal roles in osteoblast differentiation. However, the mechanisms of miRNAs regulating osteoblast mineralization still need further investigation. Here, we performed miRNA profiling and identified that miR-93 was the most significantly downregulated miRNA during osteoblast mineralization. Overexpression of miR-93 in cultured primary mouse osteoblasts attenuated osteoblast mineralization. Expression of the Sp7 transcription factor 7 (Sp7, Osterix), a zinc finger transcription factor and critical regulator of osteoblast mineralization, was found to be inversely correlated with miR-93. Then Sp7 was confirmed to be a target of miR-93. Overexpression of miR-93 in cultured osteoblasts reduced Sp7 protein expression without affecting its mRNA level. Luciferase reporter assay showed that miR-93 directly targeted Sp7 by specifically binding to the target coding sequence region (CDS) of Sp7. Experiments such as electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (ChIP), and promoter luciferase reporter assay confirmed that Sp7 bound to the promoter of miR-93. Furthermore, overexpression of Sp7 reduced miR-93 transcription, whereas blocking the expression of Sp7 promoted miR-93 transcription. Our study showed that miR-93 was an important regulator in osteoblast mineralization and miR-93 carried out its function through a novel miR-93/Sp7 regulatory feedback loop. Our findings provide new insights into the roles of miRNAs in osteoblast mineralization. © 2012 American Society for Bone and Mineral Research.