Background: Many international medical organizations recommend vitamin D supplementation for infants, especially exclusively breastfed infants. In Thailand, however, data regarding the vitamin D status in Thai infants are lacking. Such data would help to support physician decisions and guide medical practice. Methods: Full-term, exclusively breastfed infants were randomized into two groups at 2 months of age to continue exclusive breastfeeding either without vitamin D supplementation (control group, n = 44) or with vitamin D3 supplementation at 400 IU/day (intervention group, n = 43) until 6 months of age. At 6 months, the serum vitamin D (25OHD) of the infants and their mothers, serum bone marker, and infants' growth parameters were compared between the two groups. Results: The infants' serum 25OHD concentration was lower in the control group than intervention group (20.57 ± 12.66 vs. 46.01 ± 16.42 ng/mL, p < 0.01). More infants had vitamin D sufficiency (25OHD of >20 ng/mL) in the intervention group than control group (93.0% vs. 43.2%, p < 0.01). Vitamin D supplementation in breastfed infants increased the mean serum 25OHD concentration by 25.66 ng/mL (95% confidence interval, 19.07–32.25; p < 0.001) and contributed to an 88.7% decrease in the prevalence of vitamin D insufficiency/deficiency (relative risk, 0.11; 95% confidence interval, 0.04–0.35; p < 0.01).Conclusions: Most full-term, exclusively breastfed Thai infants have serum vitamin D concentration below sufficiency level at 6 months of age. However, vitamin D supplementation (400 IU/day) improves their vitamin D status and prevents vitamin D deficiency.Trial registration: The study was pre-registered in the Thai Clinical Trials Registry (TCTR20190622001) on 22/06/2019.