1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth. Although it is evident that 1,25(OH)₂D₃ inhibits growth of both estrogen receptor alpha-positive [ERα(+)] and -negative [ERα(−)] breast cancer cells, the cellular pathways contributing to these effects remain unclear. We studied the gene expression patterns in ERα(+) MCF-7 and ERα(−) MDA MB 231 human breast cancer cells following 1,25(OH)₂D₃ treatment, using cDNA expression arrays. Both cell lines showed a significant induction of the 1,25(OH)₂D₃-dependent 24-hydroxylase gene, a marker for the actions of 1,25(OH)₂D₃. In MCF-7 cells, 51 genes were up-regulated and 19 genes were down-regulated. The up-regulated genes encoded cell adhesion molecules, growth factors/modulators, steroid receptors/co-activators, cytokines, kinases and transcription factors. Of the up-regulated genes, 40% were implicated in cell cycle regulation and apoptosis and included cyclin G1 and cyclin I, p21-activated kinase-1 (PAK-1), p53, retinoblastoma like-2 [Rb2 (p130)], insulin-like growth factor binding protein-5 (IGFBP5) and caspases. Among the down-regulated genes were ERα, growth factors, cytokines and several kinases. Some of these results were confirmed by real-time PCR. In MDA MB 231 cells, 20 genes were up-regulated and 13 genes were down-regulated. Very few genes directly implicated in cell cycle regulation were up-regulated. The matrix metalloproteinases formed a major class of genes that were down-regulated in the MDA MB 231 cells. Seven genes were commonly up-regulated in both cell lines and these included transforming growth factor (TGFβ₂) and Rb2 (p130). In conclusion, the gene expression profiles of the two cell lines studied were different with a few overlapping genes suggesting that different cellular pathways might be regulated by 1,25(OH)₂D₃ to exert its growth inhibitory effects in ERα(+) and ERα(−) cells.