Objective: Our study aimed to investigate the nutritional vitamin D status of school children aged 9–15 years and white-collar workers in Zhejiang province, and evaluate the efficacy of low-dose-oral vitamin D supplementation in both populations. Methods: We conducted a prospective controlled trial during March 2014 to November 2015, comparing the efficacy of vitamin D supplements (400 IU/day) with non-intervention for 18 months in school children aged 9–15 years. Meanwhile, a before-after study was conducted among white-collar workers for 1 year. Serum 25(OH)D concentration was measured at baseline and after vitamin D supplementation, respectively. Results: At the baseline, 95% of school children and 84% of adult participants had vitamin D deficiency (<20 ng/mL). In school children, no difference was observed between the intervention and control groups with regard to anthropometric data. Serum 25(OH)D concentrations of the school children intervention group, school children control group and white-collar workers were 12.77 ± 3.01 ng/mL, 14.17 ± 3.59 ng/mL and 16.58 ± 3.66 ng/mL at baseline and increased to 17.34 ± 3.78 ng/mL, 18.04 ± 4.01 ng/mL and 17.75 ± 5.36 ng/mL after vitamin D supplementation, respectively. Although, after adjusting for potential confounders, the 400 IU oral vitamin D supplementation increased serum 25(OH)D concentration in school children (β = 0.81, p = 0.0426) as well as in white-collar workers (p = 0.0839), the prevalence of vitamin D deficiency was still very high among school children (79.23% in intervention group and 72.38% in control group) and white-collar workers (76.00%). Conclusions: High prevalence of vitamin D deficiency was common in these two study populations. Daily doses of 400 IU oral vitamin D supplementation was not able to adequately increase serum 25(OH)D concentrations. A suitable recommendation regarding the level of vitamin D supplementation is required for this Chinese population.