Genetic variants in the vitamin D receptor (VDR) gene were recently associated with asthma. The biological mechanisms explaining this association is unknown, but are likely to involve many cell types given the pleiotropic effect of its ligand, 1,25-dihydroxy-vitamin D₃ [1,25(OH)₂D₃]. Considering the prominent role of bronchial smooth muscle cells (BSMC) in the pathogenesis of asthma, experiments were conducted to explore the gene regulatory effects of 1,25(OH)₂D₃ in these cells. First, it was shown that VDR is present both at the mRNA transcript and protein levels in human BSMC. The functionality of the receptor was then demonstrated by showing a more than 200-fold change in the expression of the 24-hydroxylase (CYP24A1) gene following 1,25(OH)₂D₃ stimulation. Microarray experiments were then performed to identify differentially regulated genes and pathways in BMSC treated or not with 1,25(OH)₂D₃. A total of 729 probe sets on the U133 plus 2.0 Affymetrix GeneChip showed fold-change differences above the 1.5 threshold using the Robust Multichip Average (RMA) intensities. This corresponds to 231 unique genes that were up-regulated and 215 unique genes that were down-regulated following 1,25(OH)₂D₃ stimulation. Real-time PCR was performed to validate the microarray experiment and to confirm the regulation of asthma candidate genes. To identify the biological relevance of this regulation, biological pathways analyses were performed. The most significant network of up-regulated genes included genes involved in morphogenesis, cell growth and survival as well as genes encoding structural proteins, which are potentially involved in airway remodelling.