This study investigated the effects of altered CXCL12/CXCR4 axis on the bone morphogenetic protein 2 (BMP-2)/Smad/runt-related transcription factor 2 (Runx2)/Osterix (Osx) signal axis and osteogenic gene expression during osteogenic differentiation of mesenchymal stem cells (MSCs), to gain understanding of the link between migration and osteogenic differentiation signal axis and MSCs osteogenic differentiation mechanisms. The pHBAd-MCMV-CXCL12-GFP vector (Ad-CXCL12) was constructed and quantitative polymerase chain reaction (qPCR)/western blotting used to determine CXCL12 expression in Ad-CXCL12-transfected MSCs. MSCs were treated with Ad-CXCL12 and AMD3100 (CXCL12 inhibitor) to detect BMP-2/Smad/Runx2/Osterix expression, bone sialoprotein (BSP), osteocalcin (OCN) and osteopontin (OPN) mRNA expression, and alkaline phosphatase (ALP) activity. PCR and sequencing confirmed successful construction of Ad-CXCL12. qPCR and enzyme-linked immunosorbent assay indicated that Ad-CXCL12 transfection promoted CXCL12 expression in MSCs. At 72 hours, Runx2 and Osterix, and Smad1/5/8 mRNA and protein expressions were significantly higher in the Ad-CXCL12 group than in the control group (P < 0.01). At 1 and 2 weeks, ALP activity and BSP mRNA expression were significantly higher in the Ad-CXCL12 group than in the control group (P < 0.01), respectively. No significant difference in OCN and OPN mRNA expression was determined between Ad-CXCL12 and control groups (P > 0.05). At 3 weeks, no significant difference in mineralized nodule staining was observed between groups (P > 0.05). Changes in the CXCL12/CXCR4 migration axis affected the BMP-2/Smad/Runx2/Osterix axis and BSP, OCN and OPN mRNA expression in early-stage, but not mid-/late-stage, MSCs osteogenic differentiation, therefore affecting the ability of MSCs to undergo osteogenic differentiation.