Osteoporosis is a complex, polygenic disorder of chronic bone loss that occurs due to an imbalance between bone formation and resorption rates. As a multicellular, variable process affected by both local and systemic changes, the characterization of candidate genes associated with bone density remains problematic. Therefore, by focusing on the intrinsic genetic determinants of one cell type, the bone-forming osteoblast, the intricacies of bone mass research can be greatly reduced. Mineralization by the osteoblast has been shown to be a highly heritable phenotype, indicating a significant level of genetic regulation over its function. Focusing our genetic studies on bone formation, we have previously identified a genomic locus associated with mouse osteoblast mineralization, which lies upstream of the uncharacterized gene Zinc Finger and BTB Domain Containing 40 (Zbtb40). This gene has no known function;​ however, other members of the BTB-ZF protein family are transcription factors regulating stem cell self-renewal, differentiation, and commitment. Upon siRNA-mediated knockdown of Zbtb40 in MC3T3 mouse preosteoblasts, there was a marked reduction in differentiation capacity and function, with a complete absence of mineralized nodule formation. To further investigate these findings, a genetic mouse model was created using the CRISPR/Cas9 technology, generating a strain (Zbtb40^mut) with a mutant, truncated form of the ZBTB40 protein lacking the “BTB” protein-protein interaction domain, emulating a partial loss-of-function. Calvarial osteoblasts from Zbtb40^mut mice differentiated in culture show reduced differentiation and mineralization capabilities compared to osteoblasts from control mice. The lumbar spine of male Zbtb40^mut mice exhibited significant reductions in bone mineral density (BMD). When analyzing by Micro-Computed Tomography (μCT), it was found that trabecular bone in the L5 vertebrae from male Zbtb40^mut mice had reductions in normalized bone volume, as well as trabecular number, thickness, and connectivity compared to control mice. This data establishes Zbtb40 as a novel regulator of bone mass;​ however, the molecular characteristics remained unknown. Upon further experimental dissection, other roles appear to exist for ZBTB40 including broad transcriptional regulation, multi-lineage control of commitment, as well as regulation of proliferation, which may contribute to pathologies in bone as well as other tissues. This work has identified a novel regulator of bone mass, which may have a role in the pathogenesis of osteoporosis in humans.