This study was designed to investigate the impact of 1,25 dihydroxyvitamin D (1,25(OH)2D) on glucose metabolism during early cancer progression. Untransformed and ras-oncogene transfected (ras) MCF10A human breast epithelial cells were employed to model early breast cancer progression. 1,25(OH)2D modified the response of the ras cells to glucose restriction, suggesting 1,25(OH)2D may reduce the ras cell glucose addiction noted in cancer cells. To understand the 1,25(OH)2D regulation of glucose metabolism, following four-day 1,25(OH)2D treatment, metabolite fluxes at the cell membrane were measured by a nanoprobe biosensor, [13C6]glucose flux by 13C-mass isotopomer distribution analysis of media metabolites, intracellular metabolite levels by NMR, and gene expression of related enzymes assessed. Treatment with 1,25(OH)2D reduced glycolysis as flux of glucose to 3-phosphoglycerate was reduced by 15% (P = 0.017) and 32% (P < 0.003) in MCF10A and ras cells respectively. In the ras cells, 1,25(OH)2D reduced lactate dehydrogenase activity by 15% (P < 0.05) with a concomitant 10% reduction in the flux of glucose to lactate (P = 0.006), and reduction in the level of intracellular lactate by 55% (P = 0.029). Treatment with 1,25(OH)2D reduced flux of glucose to acetyl-coA 24% (P = 0.002) and 41% (P < 0.001), and flux to oxaloacetate 34% (P = 0.003) and 33% (P = 0.027) in the MCF10A and ras cells, respectively, suggesting a reduction in tricarboxylic acid (TCA) cycle activity. The results suggest a novel mechanism involving the regulation of glucose metabolism by which 1,25(OH)2D may prevent breast cancer progression.