The aim of this study was to investigate the acute effect of hydration status on glycemic regulation in healthy adults and explore underlying mechanisms. In this randomized crossover trial, 16 healthy adults (8 male) underwent an oral glucose tolerance test (OGTT) when hypohydrated and rehydrated, after four days of pre-trial standardization. One day pre-OGTT, participants were dehydrated for 1-h in a heat-tent with subsequent fluid restriction (HYPO) or replacement (RE). The following day, an OGTT was performed with metabolic rate measures and pre- and post-OGTT muscle biopsies. Peripheral quantitative computer tomography thigh scans were taken pre- and post-intervention to infer changes in cell volume. HYPO (but not RE) induced 1.9±1.2% body mass loss, 2.9±2.7% cell volume reduction, and increased urinary hydration markers, serum osmolality, and plasma copeptin concentration (all p≤0.007). Fasted serum glucose (HYPO 5.10±0.42 mmol∙l‑1; RE 5.02±0.40 mmol∙l-1; p=0.327) and insulin (HYPO 27.1±9.7 pmol∙L-1; RE 27.6±9.2 pmol∙L-1; p=0.809) concentrations were similar between HYPO and RE. Hydration status did not alter the serum glucose ( p=0.627) or insulin ( p=0.200) responses during the OGTT. Muscle water content was lower pre-OGTT after HYPO compared to RE (761±13 g∙kg-1 wet weight versus 772±18 g∙kg-1 RE), but similar post-OGTT (HYPO 779±15 g∙kg-1 versus RE 780±20 g∙kg‑1; time p=0.011; trial*time p = 0.055). Resting energy expenditure was similar between hydration states (stable between -1.21 and 5.94 kJ∙kg-1∙d-1; trial p=0.904). Overall, despite acute mild hypohydration increasing plasma copeptin concentrations and decreasing fasted cell volume and muscle water, we found no effect on glycemic regulation.