〔Abstract〕 Objective Investigate osteogenic differentiation of canine periodontal ligament stem cells(cPDLSCs)viaover-expression ephrinB2 in cPDLSCs. Methods cPDLSCs were isolated from the premolars and molars of Beagle. After transfected with EfnB2-GFP-Bsd and GFP-Bsd empty Vector, cPDLSCs were induced to osteogenic differentiation. Western blot was used to invest the expression of ephrinB2 protein. The effect of osteogenic differentiation of EfnB2-cPDLSCs and Vector-cPDLSCs were analyzed by RT-PCR, CCK-8, Alizarin-red S staining and ALP. Results There was no significant difference in cell proliferation between EfnB2-cPDLSCs and Vector-cPDLSCs. While EfnB2-cPDLSCs displayed an enhanced ALP activity and more prominent mineralized nodules compared with Vector-cPDLSCs. The odonto-/osteogenic genes in EfnB2-cPDLSCs were also highly enhanced. Conclusion The results of our study indicated that ephrinB2 gene-transfected cPDLSCs showed enhanced osteogenic differentiation.