OBJECTIVE—We previously reported that people prenatally exposed to famine during the Dutch Hunger Winter of 1944–1945 have higher 2-h glucose concentrations after an oral glucose tolerance test in later life. We aimed to determine whether this association is mediated through alterations in insulin secretion, insulin sensitivity, or a combination of both. RESEARCH DESIGN AND METHODS—We performed a 15-sample intravenous glucose tolerance test in a subsample of 94 normoglycemic men and women from the Dutch Famine Birth Cohort. We used the disposition index, derived as the product of insulin sensitivity and the first-phase insulin response to glucose as a measure of the activity of the β-cells adjusted for insulin resistance. In all analyses, we adjusted for sex and BMI. RESULTS—Glucose tolerance was impaired in people who had been prenatally exposed to famine compared with people unexposed to famine (difference in intravenous glucose tolerance test Kg value −21% [95% CI −41 to −4]). People exposed to famine during midgestation had a significantly lower disposition index (−53% [−126 to −3]) compared with people unexposed to famine. Prenatal exposure to famine during early gestation was also associated with a lower disposition index, but this difference did not reach statistical significance. CONCLUSIONS—Impaired glucose tolerance after exposure to famine during mid-gestation and early gestation seems to be mediated through an insulin secretion defect.