Steroid hormones including (1,25)-dihydroxyvitamin D3, estrogens, and glucocorticoids control bone development and homeostasis. We show here that the osteogenic transcription factor Runx2 controls genes involved in sterol/steroid metabolism, including Cyp11a1, Cyp39a1, Cyp51, Lss, and Dhcr7 in murine osteoprogenitor cells. Cyp11a1 (P450scc) encodes an approximately 55-kDa mitochondrial enzyme that catalyzes side-chain cleavage of cholesterol and is rate limiting for steroid hormone biosynthesis. Runx2 is coexpressed with Cyp11a1 in osteoblasts as well as nonosseous cell types (e.g. testis and breast cancer cells), suggesting a broad biological role for Runx2 in sterol/steroid metabolism. Notably, osteoblasts and breast cancer cells express an approximately 32-kDa truncated isoform of Cyp11a1 that is nonmitochondrial and localized in both the cytoplasm and the nucleus. Chromatin immunoprecipitation analyses and gel shift assays show that Runx2 binds to the Cyp11a1 gene promoter in osteoblasts, indicating that Cyp11a1 is a direct target of Runx2. Specific Cyp11a1 knockdown with short hairpin RNA increases cell proliferation, indicating that Cyp11a1 normally suppresses osteoblast proliferation. We conclude that Runx2 regulates enzymes involved in sterol/steroid-related metabolic pathways and that activation of Cyp11a1 by Runx2 may contribute to attenuation of osteoblast growth. (Molecular Endocrinology 23: 849-861, 2009)