Objectives: Defining transcriptional profiles which predict cancer cell anti-proliferative responsiveness towards 1,25-dihydroxyvitamin D₃ [1α,25(OH)₂D₃] is required to improve and tailor the chemotherapeutic application of this seco-steroid hormone to individual cancer patients. Methods: We undertook a transcriptomic approach with Affymetrix human U133 GeneChips to determine responsive and resistant gene signatures in MCF-7 breast cancer cells and 1α,25(OH)₂D₃-resistant MCF-7_Res cells, respectively. Principal component and hierarchical clustering analyses demonstrated that the patterns of responsiveness between the 2 cell types differed clearly and were used to generate heat maps. Differentially regulated gene targets were validated with Q-RT-PCR and the biological impact upon proliferation measured. Results: In untreated MCF-7_Res cells, 163 genes were up-regulated and 274 down-regulated (with a log₂ ratio of >0.5) compared to the MCF-7 controls. Using the same gene expression threshold, 1α,25(OH)₂D₃ treatment (100 nM, 6 h) of MCF-7 cells up-regulated 91 genes and down-regulated 5, whereas in MCF-7_Res, despite their resistance to the anti-proliferative effects, 156 genes were modulated with 91 being down-regulated. Strikingly, CYP24 was the only induced gene that was common to the genetic profiles of the 2 sets of 1α,25(OH)₂D₃-treated cells. Heat map analyses defined 2 sub-clusters of genes: (1) basal expression patterns associated with insensitivity towards 1α,25(OH)₂D₃ and (2) regulated expression patterns associated with 1α,25(OH)₂D₃ sensitivity. This latter cluster contained BAX, GADD45α, IGFBP-3, EGFR, MAPK4 and TGF-β₂. Time course studies confirmed the 1α,25(OH)₂D₃ regulation of TGF-β₂ in MCF-7 and non-tumourigenic MCF-12A cells but not in MCF-7_Res cells. Co-treatment of MCF-7_Res cells with exogenous TGF-β₂ plus 1α,25(OH)₂D₃ enhanced anti-proliferative and vitamin D receptor transcriptional effects. Conclusions: Basal and regulated gene patterns can be used to predict and monitor the cellular response towards vitamin D₃ compounds and may possibly be applied as a further diagnostic tool.