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To explore the critical role and function of miRNAs in the regulation of development and physiology of maxillary sinus membrane stem cell (MSMSC) osteogenesis.Microarray analysis was performed to screen the miRNAs expression profiles during the process of MSMSC osteogenic differentiation. Quantitative real-time polymerase chain reaction was applied to verify the miRNAs expression profiles. Gain- and loss-of-function experiments were used to demonstrate that miR-27b-3p inhibited MSMSC osteoblastic differentiation. Bioinformatic analysis was performed to predict the potential target of miR-27b-3p and then demonstrated by luciferase reporter assay and western blot. The negative regulation between miR-27b-3p and Sp7 was further confirmed using mimic and inhibitor of miR-27b-3p in vitro. Xenograft mice model was generated to confirm the relationship between miR-27b-3p and Sp7 using recombinant adenoviruses in vivo.MiR-27b-3p was downregulated during osteogenic differentiation of MSMSCs. The expression of Sp7, alkaline phosphatase, and osteocalcin decreased when transfected with miR-27b-3p-mimic in MSMSCs after osteogenic differentiation. MiR-27b-3p directly targeted Sp7 and inhibited the MSMSC osteogenesis in vivo.MiR-27b-3p suppressed the osteogenic differentiation of MSMSCs by directly inhibiting Sp7.