To evaluate the effectiveness of weekly vitamin D supplementation in reducing the number of acute respiratory infections (ARI) in preschool children. Randomized, double-blind, placebo-controlled trial in 303 children aged 1.5-3.5 years from 2014 to 2105 in three Chilean cities at different latitudes: Santiago (33ºS, n=101), Talcahuano (37ºS, n=103), and Punta Arenas (53ºS, n=99). Participants were allocated (1:1:1) to receive placebo, cholecalciferol (VD3) 5,600 IU/week (low-dose), or 11,200 IU/week (high-dose) for 6 months. Primary outcome was parent-reported number of ARI; secondary outcomes included number of ARI hospitalizations, change of serum 25(OH)D and LL37/cathelicidin levels, and adverse events. The mean age of participants was 26 ± 6 months; 45% were female. Baseline 25(OH)D was 24.9 ± 6.1 ng/ml, with 23% having 25(OH)D <20 ng/ml. No significant baseline clinical or laboratory differences were observed among groups. Overall, 64% (n=194) completed study participation, without baseline differences between subjects lost to follow-up versus those completing participation or differences in completion rates across groups. After 6 months, a dose-dependent increase in serum 25(OH)D was observed from the VD3 intervention (p<0.001), with a higher proportion of subjects ending the trial with 25(OH)D <20 ng/ml in the placebo group (30.8%) versus the low-dose (7.4%) and high-dose groups (5.1%). However, no group differences were observed in number of ARI (p=0.85), ARI hospitalizations (p=0.20), LL-37/cathelicidin change (p=0.30), or adverse events (p=0.41). While weekly VD3 supplementation, in doses equivalent to 800 IU and 1600 IU daily, was associated with improved 25(OH)D levels in preschoolers, we did not find a reduced number of ARI in this sample.