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Osteogenic differentiation is a complicated process that depends on various regulatory factors and signal pathways. In our research, the osteogenic differentiation capacity was analyzed by alizarin red staining, alkaline phosphatase activity, and protein levels of osteogenic differentiation markers including runt‐related transcription factor 2, bone morphogenetic protein 2, and osteocalcin (OCN). We observed a notable decrease of miR‐24‐3p level in osteogenic‐differentiated human periodontal ligament stem cells (hPDLSCs) by microarray analysis. In our gain‐ and loss‐of‐function experiments, we discovered that miR‐24‐3p has a suppression effect on hPDLSCs osteogenic differentiation. Moreover, SMAD family member 5 (Smad5), the critical osteogenic differentiation transcription factors, was predicted to be targets of miR‐24‐3p. In addition, luciferase reporter assay further proved that miR‐24‐3p directly targeted the 3′‐untranslated region of Smad5. Similarly, we found that the overexpression of miR‐24‐3p significantly decreased the Smad5 messenger RNA level in hPDLSCs, which was detected by real‐time quantitative polymerase chain reaction. Then hPDLSCs were transfected with miR‐24‐3p mimics to inhibit Smad5 expression; meanwhile, Smad5 RNA interference could significantly reverse the osteogenic differentiation inhibition effect of miR‐24‐3p. In brief, a series of data showed that miR‐24‐3p is a regulator of Smad5, playing an important role in osteogenic differentiation. Osteogenic differentiation is a complicated process that depends on various regulatory factors and signal pathways. In brief, a series of data showed that miR‐24‐3p is a regulator of SMAD family member 5 (Smad5), playing an important role in osteogenic differentiation. [ABSTRACT FROM AUTHOR]