We sought to determine whether the estrogen receptor-related receptor gamma (mEsrrg) regulated the Osteopontin (Opn) promoter through the same AP1/CAAT box element that we have previously described for mEsrra. In HeLa cells mEsrrg used an additional site present in the 5′UTR, while in ROS17/2.8 cells the AP1/CAAT site was not used, but a completely novel site surrounding the transcription start site was used. We also find that in ROS17/2.8 cells mEsrra repressed, while mEsrrg activated the Opn promoter. None of the sites identified conform to established Esrr response elements (ERREs). Additionally, the two reported mEsrrg protein isoforms showed differences in their activation potential. Mutations in the activation function 2 (AF2) of mEsrra, predicted to abolish activation, surprisingly turned mEsrra into a better activator. In contrast, similar AF2 mutations in Esrrg2 abolished its ability to activate the Opn promoter. Mutation of the DNA binding domain of mEsrra/g2 abolished transcriptional activity in HeLa and ROS17/2.8 cells. Our data indicate, first, that the two Esrr isoforms regulate Opn in a cell context-dependent manner. Second, they suggest that although the DNA binding domains of mEsrra and mEsrrg are 93% identical and required for regulation, the receptors bind to distinct Opn promoter elements, suggesting that the two isoforms may co-regulate Opn, and perhaps other genes, without competing for the same site in the promoter. Finally, the results suggest that each isoform interacts differently with co-activators and co-repressors, as highlighted by the AF2 mutation that turns mEsrra into a better activator but abolishes activity of Esrrg2. J. Cell. Biochem. 114: 2356–2362, 2013. © 2013 Wiley Periodicals, Inc.