Background: Biofortification is a strategy to relieve vitamin A (VA) deficiency. Biofortified maize contains enhanced provitamin A concentrations and has been bioefficacious in animal and small human studies. Objective: The study sought to determine changes in total body reserves (TBRs) of vitamin Awith consumption of biofortified maize. Design: A randomized, placebo-controlled biofortified maize efficacy trial was conducted in 140 rural Zambian children. The paired 13C-retinol isotope dilution test, a sensitive biomarker for VA status, was used to measure TBRs before and after a 90-d intervention. Treatments were white maize with placebo oil (VA2), orange maize with placebo (orange), and white maize with VA in oil [400 mg retinol activity equivalents (RAEs) in 214 mL daily] (VA+). Results: In total, 133 children completed the trial and were analyzed for TBRs (n = 44 or 45/group). Change in TBR residuals were not normally distributed (P , 0.0001); median changes (95% CI) were as follows: VA2, 13 (219, 44) mmol; orange, 84 (21, 146) mmol; and VA+, 98 (24, 171) mmol. Nonparametric analysis showed no statistical difference between VA+ and orange (P = 0.34); both were higher than VA2 (P = 0.0034). Median (95% CI) calculated liver reserves at baseline were 1.04 (0.97, 1.12) mmol/g liver, with 59% .1 mmol/g, the subtoxicity cutoff; none were ,0.1 mmol/g, the deficiency cutoff. The calculated bioconversion factor was 10.4 mg b-carotene equivalents/1 mg retinol by using the middle 3 quintiles of change in TBRs from each group. Serum retinol did not change in response to intervention (P = 0.16) but was reduced with elevated C-reactive protein (P = 0.0029) and a-1-acid glycoprotein (P = 0.0023) at baseline. Conclusions: b-Carotene from maize was efficacious when consumed as a staple food in this population and could avoid the potential for hypervitaminosis A that was observed with the use of preformed VA from supplementation and fortification. Use of more sensitive methods other than serum retinol alone, such as isotope dilution, is required to accurately assess VA status, evaluate interventions, and investigate the interaction of VA status and infection. This trial was registered at clinicaltrials.gov as NCT01814891. Am J Clin Nutr 2014;100:1541–50.