AIMS: To test the effect of 25(OH)D(3) (HyD) compared to vitamin D(3) on serum 25-hydroxyvitamin D levels (25(OH)D), lower extremity function, blood pressure and markers of innate immunity. METHODS: 20 healthy postmenopausal women with an average 25(OH)D level of 13.2 ng/ml (SD = ± 3.9) and a mean age of 61.5 years (SD = ± 7.2) were randomized to either 20 µg of HyD or 20 µg (800 IU) of vitamin D(3) per day in a double-blind manner. We measured on 14 visits over 4-months, 25(OH)D serum levels, blood pressure, and 7 markers of innate immunity (eotaxin, IL-8, IL-12, IP-10, MCP-1, MIP-1β, RANTES). At baseline and at 4 months, a test battery for lower extremity function (knee extensor and flexor strength, timed up and go, repeated sit-to-stand) was assessed. All analyses adjusted for baseline measurement, age and body mass index. RESULTS: Mean 25(OH)D levels increased to 69.5 ng/ml in the HyD group. This rise was immediate and sustained. Mean 25(OH)D levels increased to 31.0 ng/ml with a slow increase in the vitamin D3 group. Women on HyD compared with vitamin D(3) had a 2.8 fold increased odds of maintained or improved lower extremity function (OR= 2.79; 95%CI: 1.18-6.58), and a 5.7 mmHg decrease in systolic blood pressure (p = 0.0002). Both types of vitamin D contributed to a decrease in 5 out of 7 markers of innate immunity, significantly more pronounced with HyD for eotaxin, IL-12, MCP-1 and MIP-1 β. There were no cases of hypercalcemia at any time point. CONCLUSIONS: 20 µg of HyD per day resulted in a safe, immediate and sustained increase in 25(OH)D serum levels in all participants, which may explain its significant benefit on lower extremity function, systolic blood pressure, and innate immune response compared with vitamin D(3) . © 2011 American Society for Bone and Mineral Research.