Aim: To define the potential utility of 20-hydroxyvitamin D 3 (20(OH)D 3) as a tumorostatic agent, we assessed its in vitro antiproliferative activity and its in vivo toxicity. Materials and Methods: The antitumor activity of 20(OH)D 3 was tested against breast and liver cancer cell lines using colony formation assays. To assess in vivo toxicity, mice were injected with 5-30 μg/kg 20(OH)D 3 intraperitoneally each day for 3 weeks. Blood and organ samples were collected for clinical pathology analyses. Results: 20(OH)D 3 displays similar tumorostatic activity towards MDA-MB-453 and MCF7 breast carcinomas, and HepG2 hepatocarcinoma, in a dose-dependent manner. This compound is not hypercalcemic, does not cause detectable toxicities in liver, kidney, or blood chemistry in mice at a dose as high as 30 μg/kg. In contrast, both 25(OH)D 3 and 1,25(OH) 2D 3 caused severe hypercalcemia at a dose of 2 μg/kg. Conclusion: 20(OH)D 3 possesses high efficacy for inhibiting cancer cell proliferation in vitro and is non-toxic in vivo, supporting its further development as a potential anticancer therapeutic agent.