During periods of low dietary calcium intake, serum 1,25 dihydroxyvitamin D3 (1,25(OH)2 D) levels are elevated and regulate the bone forming osteoblast and osteocyte cells that express the vitamin D receptor. Osteocytes develop from mature osteoblasts after they have been trapped in the newly synthesized collagen matrix during bone remodeling. Early osteocytes are essential for the mineralization of the collagen matrix deposited by osteoblasts while late osteocytes are encased in mineralized tissue and act as sensors that induce bone formation in response to mechanical stress. Here we used MLO-A5 cells to assess the impact of 1,25(OH)2 D on early osteocytes that mediate matrix mineralization. MLO-A5 cells were seeded at high density and confluent cultures were cultured in osteogenic media for 6 d. At the start of osteogenic media (0 d) and in mineralizing cultures (6 d), cells were treated with vehicle, 10 nM, or 100 nM 1,25(OH)2 D for 6 or 24 h (n=4 replicates for each time point and treatment dose). At the end of treatment, cells were harvested into TriReagent and RNA was isolated in a two-step procedure (organic extraction followed by column purification). The high-quality RNA from these cultures was used for Tag-seq analysis (i.e. high throughput sequencing of the 3’ end of poly A-containing RNA species). Vitamin D-mediated differential gene expression was determined with DESeq2 and significance was assessed at the 10% FDR level. Induction of genes was affected by dose (100 nM 2-3 fold >10 nM), treatment time (24h 4-12 fold > 6h), and day in culture (0 d 3-8 fold > 6 d). Genes regulated at 6 h were mostly induced (67-88%) vs 24 h (44-52%), suggesting longer times induced secondary responses. At 6h in 0 d cells, 1,25(OH)2D up regulated genes controlling osteoblast differentiation, trabeculae morphogenesis, and dendrite formation. Genes for skeletal development, osteoblast differentiation, TGF signaling, and C/EBP signaling were also upregulated in 0 d 24 h treated cells. In addition, genes for oxidative phosphorylation and lipid/fatty acid metabolism were upregulated in 0d 24 h treated cells. At 6 d, treatment regulated genes for mineralization as well as for proteoglycan production, integrin binding, calcium signaling, and semaphoring signaling. These data show that vitamin D signaling has a significant effect on the development and function of early, mineralizing osteocytes. Supported my NIDDK award DK112365 to JCF and SC and start-up funds from UT-Austin to JCF.