To determine the dose-response of vitamin D3 supplementation on serum 25-hydroxyvitamin D [25(OH)D] among Chinese adults. In this 5-arm, randomized, double-blinded controlled trial, 76 healthy participants were assigned to orally administrate 0, 400, 800, 1200 or 2000 IU/d of vitamin D3 for 16 weeks. Serum 25(OH)D, parathyroid hormone, calcium, biomarkers of liver and renal function were measured at multiple time points. The mean (SD) serum 25(OH)D at baseline was 31.6 (8.7) nmol/L, and the dose-response relationship was curvilinear with a plateau around 6 weeks for all doses. At week 16, 25(OH)D was increased by 6.0 (6.5), 21.7 (15.8), 26.3 (12.6), 32.0 (12.8) and 36.3 (26.0) nmol/L for 0, 400, 800, 1200 and 2000 IU/d (all P a parts per thousand currency sign 0.002), corresponding to approximately 19, 53, 67, 77 and 80 % of reversion of vitamin D deficiency, respectively. Daily intake of 800 IU vitamin D3 reached a targeted 25(OH)D a parts per thousand yen 30 nmol/L in at least 97.5 % of Chinese, but not a targeted 25(OH)D a parts per thousand yen 50 nmol/L even with 2000 IU/d. Change of 25(OH)D was inversely associated with change of PTH concentration (r = -0.39, P < 0.001) after controlling for age and sex. No between-group differences were observed in terms of the change in serum calcium, alanine transaminase, aspartate aminotransferase, gamma-glutamyltransferase and creatinine (P a parts per thousand yen 0.22). Supplementation with 400, 800, 1200 or 2000 IU/d vitamin D could improve the vitamin D deficiency with various degrees. Whether 2000 IU/d vitamin D3 would generate a better result without side effect requires more studies with larger samples in future.