We previously showed that hypothalamic neuronal activity, as measured by the blood oxygen level dependent (BOLD) functional MRI signal, declines in response to oral glucose intake. To further explore the mechanism driving changes in hypothalamic neuronal activity in response to an oral glucose load, we here compare hypothalamic BOLD signal changes subsequent to an oral vs. an intravenous (i.v.) glucose challenge in healthy humans. Seven healthy normal-weight men received four interventions in random order after an overnight fast: 1) ingestion of glucose solution (75 g in 300 mL) or 2) water (300 mL); 3) intravenous infusion of 40% glucose solution (0.5 g/kg body weight, maximum 35 g) or 4) infusion of saline (0.9 % NaCl, equal volume). The BOLD signal was recorded as of 8 minutes prior to intervention (baseline) until 30 minutes after. Glucose infusion was associated with a modest and transient signal decline in the hypothalamus. In contrast, glucose ingestion was followed by a profound and persistent signal decrease, despite the fact that plasma glucose levels were almost 3-fold lower than in response to i.v. administration. Accordingly, glucose ingestion tended to suppress hunger more than i.v. infusion (P < 0.1). We infer that neural and endocrine signals emanating from the gastrointestinal tract are critical for the hypothalamic response to nutrient ingestion.