Osteoblasts have been shown to express both isoforms of estrogen receptor (ERα and ERβ). As a tool for the study of endogenous regulation of these genes the decoy strategy was employed. Human MG-63 osteoblast-like cells were transfected with a DNA decoy molecule containing a putative negative cis-element (DNA-102) located in the C distal promoter of ERα gene. Using real-time quantitative RT-PCR, we found that the DNA-102, but not scrambled DNA, produced a 36-fold increase in the level of total ERα mRNA and a 12-fold increase in the level of mRNA for the F isoform that is transcribed from the upstream F promoter, which is predominantly used in osteoblasts. This effect appears to be controlled by estrogen since 17-β-estradiol downregulated the mRNA increase. Notably, the same decoy was able to induce a 6-fold increase in ERβ mRNA transcription, indicating the coregulation of the ERα and ERβ expression. An increase in OPN but not in BMP4 expression was also observed. In addition, in decoy-treated cells, the cell growth decreased together with an increase in alkaline phosphatase activity. These findings indicated that DNA-102 decoy was able to induce a more differentiated osteoblastic phenotype. The augmentation of ERα and ERβ expression by the decoy approach may offer a further possibility for patient response to estrogenic therapy in the treatment of diseases related to estrogen deficiency.