CONTEXT Caffeine can stimulate insulin secretion by attenuating hyperglycemia in diabetes models with significant reduction of pancreatic functional β cells. Knowledge of these mechanisms could contribute to new strategies for treating diabetes. OBJECTIVE This study evaluated the effects of caffeine and physical exercise on glycemic and insulin responses in diabetic rats. MATERIALS AND METHODS The diabetes model was induced by intraperitoneal administration of 60 mg/kg of streptozotocin (STZ). Animals were divided into six groups: control, caffeine, STZ control, STZ caffeine, STZ sulfonylurea, and STZ caffeine + sulfonylurea. Acutely, control animals received 6 mg of caffeine and 10 mg/kg sulfonylurea or 10 mg/kg saline. Animals were sacrificed after physical exercise; blood samples were collected for glucose, glycerol, lactate, and insulin analyses. Cardiovascular responses were recorded before and after treatments. A one-way ANOVA and the post hoc Student-Newman-Keuls test were used to analyze statistical differences between treatments (p < 0.05). RESULTS About 6 mg/kg of caffeine did not alter cardiovascular responses, but promoted blood glucose reduction after 60 min of exercise when compared to animals in the control groups (387-187 mg/dL; p < 0.05). Insulin levels increased significantly (0.6-10 µU/mL; p < 0.05) in rats that received acute caffeine treatment associated with sulfonylurea compared to rats in the other groups. DISCUSSION AND CONCLUSION Acute caffeine intake with exercise can increase glucose uptake enhancing insulin secretion stimulated by sulfonylurea in β cells-deficient pancreas. The results indicate the potential use of caffeine as a strategy for glycemic and insulin control in diabetes.