We previously demonstrated that the orphan nuclear receptor, estrogen receptor-related receptor alpha (ERRalpha) is highly expressed in osteoblasts and osteoclasts, regulates osteogenesis and expression of osteoblast-associated markers in the rat calvaria cell differentiation system, and is dysregulated in the rat ovariectomy model of postmenopausal osteoporosis. There are conflicting published data on the transcriptional regulation by ERRalpha of the gene for osteopontin (OPN), an extracellular matrix protein required in bone remodeling, and a potential direct target mediating ERRalpha effects in bone. We therefore readdressed OPN gene regulation by ERRalpha in both osteoblastic (rat osteosarcoma ROS17/2.8 cells) and non-osteoblastic (HeLa) cell lines using a mouse proximal 2 kb OPN promoter fragment. A minimal OPN promoter fragment spanning from -56 to +9 bp is activated in HeLa cells but repressed it in ROS17/2.8 cells. Adenine scanning mutagenesis revealed the presence of a non-canonical ERRalpha response element in this minimal promoter. Surprisingly, prototypical inactivating mutations in the activation function 2 (AF2) domain or a naturally occurring allelic variant of ERRalpha (ERRalphaH408) were all better activators than wild-type ERRalpha in HeLa cells, activities that were generally paralleled by repression in ROS17/2.8 cells. Finally, we found that the N-terminus of ERRalpha harbors a repressor domain that acts in a cell context-dependent manner. We conclude that OPN is an ERRalpha target gene whose promoter is regulated by ERRalpha in a cell context-dependent manner and that a predicted silencing mutation in AF2 or a more flexible helix 12 increases ERRalpha transcriptional activity, effects with implications for ERRalpha as a therapeutic target in bone.