The paper explored the regulatory role of oligodeoxynucleotides (ODNs) with specific sequences in the proliferation and activation of osteoblast, using human osteoblast-like cell line MG 63 as the model. Through the administration of ODNs to MG 63 cells at a concentration of 1.0 μg/mL, ODN MT01 with positive effects on proliferation and activation of osteoblast was selected from 11 different ODNs by methyl thiazolyl tetrazolium (MTT) assay and alkaline phosphatase (ALP) activity measurement. To get a deeper insight into the molecular mechanism, effects of ODN MT01 treatment on the expression level of Sp7, runx-2, collagen-I, osteoprotegerin (OPG) and RANK ligand (RANKL) were determined using quantitative real time PCR and Western blotting. Remarkably, the mRNA and protein expression levels of Sp7, runx-2, collagen-I and OPG were improved after ODN MT01 treatment. Meanwhile, the protein expression level of RANKL was dramatically decreased. These results suggested that ODN MT01 had a significant impact in facilitating osteogenic proliferation and activation, and provided a direct evidence for the notion that single strand ODN could regulate the balance of bone formation and resorption, and thus was of great potential in the rebuilding of alveolar bone.