The vitamin D endocrine system is essential for calcium metabolism and skeletal integrity. 1,25-dihydroxyvitamin D [1,25(OH)D] regulates bone mineral homeostasis and acts directly on osteoblasts. In the present study we characterized the transcriptional regulation of the class 3 semaphorin (Sema3) gene family by 1,25(OH)D in osteoblastic cells. Class 3 semaphorins are secreted proteins that regulate cell growth, morphology and migration, and were recently shown to be involved in bone homeostasis. In ST2, MC3T3-E1 and primary calvarial osteoblast cell cultures we found that all members of the Sema3 gene family were expressed, and that Sema3e and Sema3f were the most strongly induced 1,25(OH)D target genes among the studied cell types. In addition, transcription of Sema3b and Sema3c was upregulated, whereas Sema3d and Sema3g was downregulated by 1,25(OH)D in different osteoblastic cells. Chromatin immunoprecipitation analysis linked to DNA sequencing (ChIP-seq analysis) revealed the presence of the vitamin D receptor at multiple genomic loci in the proximity of Sema3 genes, demonstrating that the genes are primary 1,25(OH)D targets. Furthermore, we showed that recombinant SEMA3E and SEMA3F protein were able to inhibit osteoblast proliferation. However, recombinant SEMA3s did not affect ST2 cell migration. The expression of class 3 semaphorins in osteoblasts together with their regulation by 1,25(OH)D suggests that these genes, involved in the regulation of bone homeostasis, are additional mediators for 1,25(OH)D signaling in osteoblasts.