The aim of the present study was to investigate the effects of quercetin on the mitogen-activated protein kinase (MAPK) signaling pathway in the osteogenic differentiation of rat mesenchymal stem cells (MSCs). A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and an alkaline phosphatase (ALP) assay were used to determine the effects of quercetin (concentrations of 0.1, 1 and 10 µmol/l) on the proliferation and differentiation of MSCs and the expression of ALP, respectively. In addition, through the introduction of inhibitors of p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and c-Jun NH2-terminal kinase (JNK), the effects of quercetin on the proteins, ALP, collagen type I (COL I) and bone γ-carboxyglutamate protein (BGP), which are indicators of osteogenic differentiation, were investigated. Immunoblotting was performed to determine the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, while fluorescent quantitative polymerase chain reaction was used to determine the mRNA expression levels of transforming growth factor (TGF)-β1, bone morphogenetic protein (BMP)-2 and core binding factor (CBF)α1. At all the concentrations tested, the concentrations of 10, 1 and 0.1 µmol/l quercetin were shown to promote the differentiation of MSCs and the expression of ALP, in which the concentration of 10 µmol/l was optimal. When compared with the control group, the phosphorylation levels of p38 MAPK, ERK1/2 and JNK, the protein expression levels of ALP, COL I and BGP, and the mNRA expression levels of TGF-β1, BMP-2 and Cbfα1 were increased in the quercetin-treated group. However, with the introduction of inhibitors, the levels of phosphorylated p38 MAPK, ERK1/2 and JNK, and the protein expression levels of ALP, COL I and BGP decreased. Furthermore, the mRNA expression levels of TGF-β1, BMP-2 and CBFα1 decreased in the quercetin + SP600125 (inhibitor of JNK) and quercetin + PD98059 (inhibitor of ERK1/2) groups. Therefore, quercetin was demonstrated to promote the osteogenic differentiation of MSCs by activating the MAPK signaling pathway. The ERK1/2 and JNK signaling pathways regulate the expression of TGF-β1, BMP-2 and CBFα1. Thus, activation of the ERK1/2 and JNK signaling pathways may play a leading role in the quercetin-promoted osteogenic proliferation and differentiation of MSCs.