Abstract Context Glucose homeostasis is under circadian control through both endocrine and intracellular mechanisms, with several lines of evidence suggesting that melatonin affects glucose homeostasis. Objective To evaluate the acute in vivo and in situ effects of melatonin on secretion of the incretin hormones, glucagon-like-peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), and their impact on β-cell insulin secretion. Design A human randomized, double-blinded, placebo-controlled crossover study combined with a confirmatory in situ study of perfused rat intestines. Setting Aarhus University Hospital. Methods Fifteen healthy male participants were examined 2 × 2 times: an oral glucose tolerance test (OGTT) was performed on day 1 and an isoglycemic IV glucose infusion replicating the blood glucose profile of the OGTT day was performed on day 2. These pairs of study days were repeated on treatment with melatonin and placebo, respectively. For the in situ study, 6 rat intestines and 4 rat pancreases were perfused arterially with perfusion buffer ± melatonin. The intestines were concomitantly perfused with glucose through the luminal compartment. Results In humans, melatonin treatment resulted in reduced GIP secretion compared with placebo (ANOVA P = 0.003), an effect also observed in the perfused rat intestines (ANOVA P = 0.003), in which GLP-1 secretion also was impaired by arterial melatonin infusion (ANOVA P < 0.001). Despite a decrease in GIP levels, the in vivo glucose-stimulated insulin secretion was unaffected by melatonin (P = 0.78). Conclusion Melatonin reduced GIP secretion during an oral glucose challenge in healthy young men but did not affect insulin secretion. Reduced GIP secretion was confirmed in an in situ model of the rat intestine.