1α,25-dihydroxyvitamin D₃ (calcitriol), an active form of vitamin D₃ metabolite, can act by several pathways, including via genomic receptor mediated by the VDR. It can regulate bone mineral homeostasis, cell proliferation, and cell differentiation and also plays an important role on tumor promotion and metastasis. To examine the effects of calcitriol and its analogs on cell proliferation, osteopontin (OPN) protein, and mRNA expression in three different bone cell lines (ROS 17/2.8, MC3T3-E1, and MG-63), we treated cells with various doses of calcitriol or its analogs (calcipotriol (BT), 25-OH-16-ene-23-yne-D₃ (AT), 25(OH)D₃ (BO), and 24,25(OH)2D₃ (AS)) (10^-8–10^-10 M). In MC3T3-E1 cells, all analogs had an inhibitory effect from day 3 to day 7, except for 3 days of 0.1, 0.01 nM calcitriol treatment;​ 5 days of 1, 10 nM BO;​ and 7 days of 0.01 nM AT and BO treatment. In ROS 17/2.8 cells, there were no significant differences between control and all calcitriol analogs (all four concentrations for 7 days, and 0.01, 1, 10 nM treatment for 5 days). However, in MG-63 cells, 10 nM of calcitriol and analogs treatment had significant inhibitory effects on cell proliferation from day 1 to day 7, except 10 nM BT and AT treatment on day 5. For the lower concentration of 0.01 NM, there were no significant differences between control and all calcitriol analogs from day 1 to day 7, except AS on day 1 and AT, BO, and AS on day 3. TCA precipitation after metabolic labeling with ³²PO₄ or ³⁵S-methionine showed that calcitriol, BT, and AT increased the level of total phosphorylated protein in ROS 17/2.8 and MC3T3-E1 cells, but vitamin D analogs did not affect the total amount of protein secreted by these two cell lines. Immunoadsorption assays to assess OPN protein expression showed that compared to control cells, calcitriol, and BT but not AT, BO, or AS stimulated the expression of OPN protein. Northern blots demonstrated that only calcitriol and BT increased OPN mRNA expression in both ROS 17/2.8 and MC3T3-E1 cells. Data from transfection assay showed that the induction of OPN expression by calcitriol and BT in both ROS 17/2.8 and MC3T3-E1 cells is likely due to transcriptional regulation. Calcitriol and BT also stimulated osteocalcin (OCN) mRNA expression in ROS 17/2.8 cells. In MG-63 cells, we detected no phosphorylated protein, including OPN, but calcitriol, BT, and AT increased the total secreted protein. Northern blot analyses of total RNA or mRNA detected no secreted OPN mRNA in cells $\pm$ treatment with calcitriol or its analogs after treatment of different cell densities or different time courses. Southern blot analysis indicated that the OPN gene was present, and a putative OPN mRNA was detected. Calcitriol, BT, and AT inhibited VDR mRNA expression in MG-63 cells but also stimulated osteocalcin (OCN), alkaline phosphatase (ALP), type I collagen (COLI) protein expression, and COLI mRNA expression in MG-63 cells. These findings suggest that some analogs of calcitriol can stimulate genomic and nongenomic pathways in osteoblast-like cells but that effects vary with cell line. However, MG-63 cells may not be a good model of osteoblast development in one critical aspect, the production of OPN.