Background: The present study aimed to determine the role and mechanism of miR-23 with respect to regulating the osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs). Materials: The expression of miR-23 and MEF2C was measured in osteoporosis (OP) patients and healthy controls by a quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR). The correlation between miR-23 and MEF2C was determined by the Pearson correlation coefficient. Moreover, bioinformatic analysis was performed using public databases. Target gene function and potential pathways were further examined. Then, we used a miR-23 mimic or inhibitor to further explore the potential mechanism of miR-23. Results: miR-23 is found to be up-regulated and MEF2C is down-regulated in OP patients compared to healthy controls. miR-23 had a negative correlation with MEF2C (r = −0.937, p = 0.001). Bioinformatic analysis revealed that a total of 664 overlapping target genes were found in the TargetScan (http://www.targetscan. org), miRDB (http://mirdb.org) and miRanda (http://www.microrna.org/microrna/home.do) databases. Moreover, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that miR-23 may regulate the mitogan-activated protein kinase (MAPK) signaling pathway. miR-23 is down-regulated and MEF2C is significantly up-regulated in the osteogenic differentiation of hBMSCs. MEF2C was significantly up-regulated in the osteogenic differentiation of hBMSCs. Overexpression of miR-23 significantly down-regulated alkaline phosphatase (ALP) activity and calcium deposition, whereas the miR-23 inhibitor had the opposite effects. Moreover, overexpression of miR-23 significantly decreased osteoblastrelated markers (Runx2, Osx, ALP and OCN). Further experiments confirmed that MEF2C is a direct target of miR-23. Moreover, the miR-23 mimic enhanced the expression of p-p38 but had no effect on p-JNK. Conclusions: miR-23 decreases the osteogenic differentiation of hBMSCs through the MEF2C/MAPK signaling pathway.