The vitamin D endocrine system is essential for calcium and phosphate homeostasis and skeletal mineralization. 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone binds to the vitamin D receptor (VDR) to regulate gene expression. In turn, these gene products mediate the actions of 1,25(OH)2D3 in mineralregulating target cells. 1,25(OH)2D3 impacts bone indirectly by promoting intestinal absorption of calcium and phosphate and directly by acting on osteoblasts and osteoclasts. Despite the direct regulatory roles of 1,25(OH)2D3 in bone, relatively little is known about the mechanisms or 1,25(OH)2D3-target genes in skeletal cells. Here, we identified meningioma 1 (MN1) and semaphorin 3B (SEMA3B) as two novel 1,25(OH)2D3-stimulated genes in osteoblastic cells, and uncovered their new functions in skeletal physiology. We demonstrated that MN1 is a coactivator for VDR-mediated transcription, and calvarial osteoblasts derived from MN1 knockout mice displayed altered morphology, decreased growth rate, impaired motility, attenuated 1,25(OH)2D3/VDR-mediatedtranscription, reduced alkaline phosphatase activity, decreased mineralized nodule formation, but enhanced adipogenesis. In addition, MN1 knockout osteoblasts are defective in supporting 1,25(OH)2D3-stimulated osteoclastogenesis, presumably due to marked reduction in the RANKL:OPG ratio. These data indicate an important role for MN1 in maintaining appropriate osteoblast proliferation, maturation and function. This may partially account for the intramembranous ossification defects of cranial bones in MN1 knockout mice. Our data reveal that osteoblast-derived SEMA3B alters the global skeletal homeostasis in intact animals and the bone cell activities in cultures. Transgenic mice with osteoblast-targeted over-expression of SEMA3B develop osteopenia, with decreased body weight, reduced bone mineral density, and aberrant trabecular structure compared to the nontransgenic littermates. Histomorphometry studies indicated that this was likely due to increased osteoclast numbers and activity. Indeed, primary osteoblasts obtained from SEMA3B transgenic mice stimulated osteoclastogenesis to a greater extent than nontransgenic osteoblasts. This study revealed the novel roles of SEMA3B for controlling bone cell activities in vitro and bone remodeling in vivo. Collectively, we characterized MN1 and SEMA3B as 1,25(OH)2D3-induced genes in osteoblasts and established their significance in regulating osteoblast function, osteoclast formation, and skeletal homeostasis. This provides further insights about the direct actions of 1,25(OH)2D3 in bone remodeling.