Regular exercise improves the cerebrovascular function and has shown considerable therapeutic effects on a wide variety of brain diseases. However, the influence of exercise on different aspects of the cerebrovascular function remains to be comprehensively examined. In this study, we combined awake-brain photoacoustic microscopy (PAM) and a motorized treadmill to assess the effects of both acute exercise stimulation and endurance exercise training on the cerebrovascular function and cerebral oxygen metabolism under both physiological and pathological conditions. Acute exercise stimulation in nondiabetic mice resulted in robust vasodilation, increased cerebral blood flow (CBF), reduced oxygen extraction fraction (OEF), and unchanged cerebral metabolic rate of oxygen (CMRO2)-demonstrating the utility of this experimental setting to evaluate the cerebrovascular reactivity. Also, endurance exercise training for six weeks in diabetic mice reversed the diabetes-induced increases in the resting-state CBF and CMRO2 and maintained a stable OEF and CMRO2 under the acute exercise stimulation-shedding new light on how exercise protects the brain from diabetes-induced small vessel disease. In summary, we established an experimental approach to assess the effects of both acute exercise stimulation and endurance exercise training on the cerebrovascular function and tissue oxygen metabolism at the microscopic level and applied it to study the therapeutic benefits of endurance exercise training in diabetic mice.