Vitamin D (VD) deficiency (serum 25(OH)D < 50 nmol/L) affects 27.3% of preschool children in Mexico. The purpose of this study was to assess the effect of vitamin D supplementation at different doses on serum 25(OH)D concentrations in preschool children. In a randomized control trial, 222 children 12–30 months old were randomly assigned to one of four treatment groups: (1) Vitamin D2 (Ergocalciferol) 400 IU/day (n= 56); (2) Vitamin D2 (Ergocalciferol) 800 IU/day (n= 55); (3) Vitamin D3 (Cholecalciferol) 1000 IU/day (n= 56); or (4) multiple micronutrients (MM) non-VD (n= 55). Supplements were given five days/wk for three months. Serum 25(OH)D was measured at baseline and after three months. At baseline, mean serum 25(OH)D was 58.9 ± 12.6 nmol/L and 23.4% were VD-deficient. There was a statistically significant increase in serum concentrations of 25(OH)D (range across groups: +8.2 to +17.3 nmol/L). Additionally, the prevalence of vitamin D deficiency decreased after three months: for D2 400 IU, −9.0%; for D2 800 IU, −11.0%; for D3 1000 IU, −18.0%; and for MM non-VD, −2.8% (p< 0.05). No adverse effects were observed. VD supplementation for three months was effective for increasing serum 25(OH)D concentrations and for reducing VD deficiency in preschool children. The highest efficacy was observed by giving 1000 IU D3/d.