Objectives: To compare the efficacy of cholecalciferol and ergocalciferol in raising 25-hydroxyvitamin D (25(OH)D) level in Thai female healthcare workers. Methods: A randomized control trial was conducted in healthy female healthcare workers. Randomization allocated the participants into vitamin D2 group (N = 43), receiving ergocalciferol 20,000 IU weekly and vitamin D3 group (N = 40), receiving cholecalciferol 1000 IU daily for 12 months. Venous blood sample was collected at baseline, 6 and 12 months for serum 25(OH)D, parathyroid hormone and calcium. Compliance was also assessed. Results: The mean age of the participants was 50.6 ± 9.9 and 50.9 ± 8.4 years in vitamin D2 and D3 groups (P = 0.884). The mean 25(OH)D levels were 16.91 ± 6.07 ng/mL and 17.62 ± 4.39 ng/mL (P = 0.547), respectively. Both groups had significant improvement in 25(OH)D level at 6 months (from 16.91 ± 6.07 to 21.67 ± 5.11 ng/mL and 17.62 ± 4.39 to 26.03 ± 6.59 ng/mL in vitamin D2 and D3 group). Improvement was significantly greater with cholecalciferol (P = 0.018). The level plateaued afterwards in both groups. Only cholecalciferol could increase 25(OH)D in participants without vitamin D deficiency (6.88 ± 4.20 ng/mL increment). Compliance was significantly better in vitamin D2 group (P = 0.025). Conclusions Daily cholecalciferol supplementation resulted in a larger increase in serum 25(OH)D level during the first 6 months comparing to weekly ergocalciferol. While vitamin D3 could increase serum 25(OH)D level in all participants, vitamin D2 could not do so in participants without vitamin D deficiency.