In a randomized crossover trial, 75 young adults (healthy weight, overweight or with obesity) consumed a drink containing sucralose, sweetness-matched sucrose or water. We show that acute consumption of sucralose versus sucrose stimulates hypothalamic blood flow (P < 0.018) and greater hunger responses (P < 0.001). Sucralose versus water also increases hypothalamic blood flow (P < 0.019) but produces no difference in hunger ratings. Sucrose, but not sucralose, increases peripheral glucose levels, which are associated with reductions in medial hypothalamic blood flow (P < 0.007). Sucralose, compared to sucrose and water, results in increased functional connections between the hypothalamus and brain regions involved in motivation and somatosensory processing. These findings suggest that non-caloric sweeteners could affect key mechanisms in the hypothalamus responsible for appetite regulation.