1,25-dihydroxyvitamin D₃ (1,25D or calcitriol), the active form of vitamin D₃ (VitD), exhibits anticancer actions in various experimental models of breast cancer (BCa) and prostate cancer (PCa). However, the activity of dietary VitD must be tested in in vivo models of cancer. Since CYP27B1, the 1a-hydroxylase that catalyzes synthesis of 1,25Dformation in the kidney, is also expressed in normal and malignant breast and prostate cells, we hypothesize that dietary VitD will be converted to 25(OH)D in the liver and then to 1,25Dlocally in the cancer microenvironment, where it will exert paracrine anticancer actions. In recent experiments we found that nude mice bearing MCF-7 BCa xenografts exhibited significant tumor shrinkage (>50%) following ingestion of a VitD-supplemented diet (5000 IU/kg) compared to a control diet (1000 IU/kg). Dietary VitD caused tumor inhibition that was equivalent to calcitriol administered as intraperitoneal injections at 0.025, 0.05 or 0.1 µg/mouse 3x week. Dietary vitD also inhibited PC-3 PCa xenograft growth, but to a lesser extent than MCF-7 tumors. At the 0.05 µg and 0.1 µg doses calcitriolcaused modest but significant elevations in serum calcium levels indicating that the dietary VitD comparison was to a maximally safe calcitriol dose. However, dietary VitDdid not increase serum calcium, demonstrating its safety at the concentration tested. The tumor-bearing mice receiving the VitD-supplemented diet showed elevated circulating 1,25Dlevels, no change in renal CYP27B1 mRNA and increased CYP27B1 mRNA in both BCa and PCa tumors, suggesting that extra-renal sources including the tumors contributed to the elevated circulating 1,25D concentration. Mice without tumors receiving the same VitD-supplemented diet did not show an increase in serum 1,25Dlevels. Both 1,25Dand dietary VitD were equivalent in suppressing estrogen synthesis and signaling in mice with BCa xenografts and inhibiting other pro-inflammatory and growth signaling pathways in mice with both BCa and PCa xenografts. These preclinical data demonstrate the potential utility of dietary VitD supplementation in cancer therapy and prevention. These findings also suggest that extra-renal synthesis plays an important role in elevating serum 1,25Dlevels in tumor-bearing mice.