There is an inverse correlation between exposure to sunlight (the major source of vitamin D) and the risk for prostate cancer, the most common noncutaneous cancer and second most common cause of death from cancer in American men. The active metabolite of vitamin D, 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] acting through the vitamin D receptor decreases prostate cancer cell growth and invasiveness. The precise mechanisms by which 1,25(OH)(2)D(3) inhibits growth in prostate cancer have not been fully elucidated. Treatment with 1,25(OH)(2)D(3) causes an accumulation in the G(0)/G(1) phase of the cell cycle in several prostate cancer cell lines. One potential target known to regulate the G(0)/G(1) to S phase transition is c-Myc, a transcription factor whose overexpression is associated with a number of cancers including prostate cancer. We find that 1,25(OH)(2)D(3) reduces c-Myc expression in multiple prostate epithelial cell lines, including C4-2 cells, an androgen-independent prostate cancer cell line. Reducing c-Myc expression to the levels observed after 1,25(OH)(2)D(3) treatment resulted in a comparable decrease in proliferation and G(1) accumulation demonstrating that down-regulation of c-Myc is a major component in the growth-inhibitory actions of 1,25(OH)2D(3). Treatment with 1,25(OH)(2)D(3) resulted in a 50% decrease in c-Myc mRNA but a much more extensive reduction in c-Myc protein. Treatment with 1,25(OH)(2)D(3) decreased c-Myc stability by increasing the proportion of c-Myc phosphorylated on T58, a glycogen synthase kinase-3beta site that serves as a signal for ubiquitin-mediated proteolysis. Thus, 1,25(OH)(2)D(3) reduces both c-Myc mRNA levels and c-Myc protein stability to inhibit growth of prostate cancer cells.