The daily supplementation of vitamin D is mandatory for infants. However, there are still conflicting opinions about the exact daily dose. Thus, we aimed to evaluate a daily supplementation dose of 200 IU is sufficient and compared the supplementation doses of 200 and 400 IU per day. One hundred and sixty-nine infants were randomly assigned to two groups (group 1, 200 IU/day; group 2, 400 IU/day) and there were 75 infants in group 1 and 64 were in group 2 with a total number of 139. The median levels of 25-hydroxyvitamin D3 were significantly increased in group 2 at the age of 4 months (group 1, 39.60 mcg/L; group 2, 56.55 mcg/L; p < 0.0001). We clearly demonstrated that at the age of 4 months, none of the infants on the group 2 had a serum level of 25-hydroxyvitamin D3 less than 30 mcg/L. However, 21.3% of the infants in group 1 had a level below 30 mcg/L. Thus, in order to avoid vitamin D deficiency and rickets, we recommend supplementation dose of vitamin D at 400 IU/day as a safe and effective dose.