MicroRNAs are identified as negative regulators in gene expression through silencing gene expression at the post-transcriptional and translational levels. Bone morphogenetic protein 9 (BMP9) is the most effective in inducing osteogenesis in the BMP family, the members of which were originally identified as osteoinductive cytokines. In the current study, the role of miR‑23b in the progression of BMP9‑induced C2C12 myoblasts was investigated. The results indicated that miR‑23b was significantly downregulated in C2C12 myoblasts induced by BMP9. Overexpression of miR‑23b significantly inhibited osteogenesis in the C2C12 myoblasts. In addition, it was observed that Runx2 was negatively regulated by miR‑23b at the post‑transcriptional level, via a specific target site within the 3'UTR of Runx2. Knockdown of Runx2 promoted miR‑23b‑induced inhibition of osteogenesis in C2C12 myoblasts. The expression of Runx2 was observed to be frequently upregulated in osteoblast cell lines and inversely correlated with miR‑23b expression. Thus, the results of the present study suggest that miR‑23b inhibits BMP9‑induced C2C12 myoblast osteogenesis via targeting of the Runx2 gene, acting as a suppressor. The current study contributes to the understanding of the functions of BMP9 in ossification.