Protracted periods of increased cortisol production, as may be seen in an acute illness, may lead to transient hyperglycemia. Increasing evidence suggests that cortisol may then mediate the development of insulin resistance and potentially lead to the development of overt diabetes. Evidence in animal studies also suggests that under conditions of stress Ginkgo biloba extract could reduce plasma cortisol production and so the primary aim of this study was to determine the effect of Ginkgo biloba extract ingestion on plasma cortisol production during an acute period of glucose challenge. Healthy non-diabetic, glucose tolerant volunteers (n = 30, (10/20, M/F); age, 45.7 ± 9.9 years old) completed a randomized, double-blind, placebo-controlled crossover study when they ingested Ginkgo biloba extract (120 mg/day as a single dose) and placebo during each 3-month arm. A standard 75 g oral glucose tolerance test was performed at the end of each cycle and blood was collected and used to measure plasma glucose, insulin, c-peptide and cortisol. Fasting plasma cortisol was significantly lower after the Ginkgo biloba cycle than the placebo cycle (326 ± 149 vs. 268 ± 121 nmol/L, respectively; p = 019). The plasma cortisol area under the curve during the 2-hour test (AUC_0-2) was also significantly lower after ingestion of the Ginkgo biloba cycle compared to the placebo (668 ± 265 vs. 530 ± 213 nmol/L/h, respectively; p < 0.001). It is concluded that the ingestion of Ginkgo biloba extract has effect on the hypothalamic-pituitary-adrenal axis leading to reduced basal cortisol levels and reduced cortisol production in response to acute hyperglycemic challenge.