The V-ATPased2 protein constitutes an important subunit of the V-ATPase proton pump, which regulates bone homeostasis; however, currently little is known about its transcriptional regulation. Here, in an attempt to understand regulation of the V-ATPase d2 promoter, we identified the presence of NFATc1, microphthalmia-associated transcription factor (MITF)- and myocyte enhancer factor 2 (MEF2)-binding sites within the V-ATPase d2 promoter using complementary bioinformatic analyses, chromatin immunoprecipitation, and electromobility shift assay. Intriguingly, activation of the V-ATPase d2 promoter by NFATc1 was enhanced by either MEF2 or MITF over-expression. By comparison, coexpression of MITF and MEF2 did not further enhance V-ATPase d2 promoter activity above that of expression of MITF alone. Consistent with a role in transcriptional regulation, both NFATc1 and MITF proteins translocated from the cytosol to the nucleus during RANKL-induced osteoclastogenesis, whereas MEF2 persisted in the nucleus of both osteoclasts and their mononuclear precursors. Targeted mutation of the putative NFATc1-, MITF-, or MEF2-binding sites in the V-ATPase d2 promoter impaired its transcriptional activation. Additionally retroviral overexpression of MITF or MEF2 in RAW264.7 cells potentiated RANKL-induced osteoclastogenesis and V-ATPase d2 gene expression. Based on these data, we propose that MEF2 and MITF function cooperatively with NFATc1 to transactivate the V-ATPase d2 promoter during RANKL-induced osteoclastogenesis. © 2009 by The American Society for Biochemistry and Molecular Biology, Inc.