Context: Intrauterine exposure to elevated glucose concentrations may be a mediating factor in prenatal programming of offspring diabetes risk. However, studies examining the effects of maternal glucose concentration on measures of insulin sensitivity and β-cell response in prepubertal children are limited. Objective: We tested the hypothesis that maternal gestational glucose concentration would be inversely associated with children's insulin sensitivity independent of adiposity and positively associated with children's β-cell response independent of adiposity and insulin sensitivity. Design, Setting, and Participants: This was a cross-sectional study of 21 children aged 5-10 yr in a clinical research setting. Outcomes Measured: Children's insulin sensitivity index and basal, static, dynamic, and total β-cell response to glucose were determined by mathematical modeling using insulin, glucose, and C-peptide values after a liquid meal tolerance test. Children's percent body fat was determined by dual-energy x-ray absorptiometry. Maternal gestational glucose concentration for the target pregnancy was determined after a 50-g, 1-h oral glucose challenge test at 24-28 wk gestation. Results: Maternal glucose concentration was significantly, inversely associated with children's insulin sensitivity, independent of percent fat and ethnicity (P <0.05). A significant, positive association was observed for maternal glucose concentration with static β-cell response, independent of percent fat and insulin sensitivity (P < 0.05). Conclusions: Maternal gestational glucose concentration was significantly associated with offspring insulin sensitivity and β-cell response independent of adiposity. These results suggest that maternal glucose may program the fetus both at the pancreas and at the level of insulin target tissues such as skeletal muscle and liver. Copyright © 2011 by The Endocrine Society.