In a prior randomized trial, we found that the incidence of influenza A was less in the vitamin D3 group than among those on placebo, but the total incidence of either influenza A or B did not differ between groups. In this trial, the incidence of influenza A or B was less in the vitamin D3 group than in the placebo group only during the first half of the study. To elucidate whether vitamin D3 has preventive actions against influenza A, we conducted another trial during the 2009 pandemic of the H1N1 subtype of influenza A. Students (n = 247) of a Japanese high school were randomly assigned to receive vitamin D3 supplements (n = 148; 2000 IU per day) or a placebo (n = 99) in a double-blind study for 2 months. The primary outcome was incidence of influenza A diagnosed by a rapid influenza diagnostic test by medical doctors. Influenza A was equally likely in the vitamin D3 group (20/148: 13.5%) compared with the placebo group (12/99: 12.1%). By post hoc analysis, influenza A occurred significantly less in the vitamin D3 group (2/148: 1.4%) compared with the placebo group (8/99: 8.1%) (risk ratio, 0.17; 95% confidence interval, 0.04 to 0.77; P = 0.009) in the first month. However, during the second month, the vitamin D3 group experienced more events and effectively caught up with the placebo group. Vitamin D3 supplementation did not lower the overall incidence of influenza A during the 2009 H1N1 pandemic. A post hoc analysis suggests that the initial benefit during the first month of treatment was lost during the second month.