Calcitriol (1,25-dihydroxyvitamin D(3)) inhibits the growth and stimulates the differentiation of prostate cancer (PCa) cells. The effects of calcitriol are varied, appear to be cell-specific and result in growth arrest and stimulation of apoptosis. Our goal was to define the genes involved in the multiple pathways mediating the anti-proliferative effects of calcitriol in PCa. We used cDNA microarray analysis to identify calcitriol target genes involved in these pathways in both LNCaP human PCa cells and primary prostatic epithelial cells. Interestingly, two of the target genes that we identified play key roles in the metabolism of prostaglandins (PGs), which are known stimulators of PCa cell growth and progression. The expression of the PG synthesizing cyclooxygenase-2 (COX-2) gene was significantly decreased by calcitriol, while that of PG inactivating 15-prostaglandin dehydrogenase gene (15-PGDH) was increased. We postulate that this dual action of calcitriol would reduce the levels of biologically active PGs in PCa cells decreasing their proliferative stimulus and contribute to the growth inhibitory actions of calcitriol. In addition, we propose that calcitriol can be combined with non-steroidal anti-inflammatory drugs that inhibit COX activity, as a potential therapeutic strategy to improve the potency and efficacy of both drugs in the treatment of PCa.