Capillaries were believed to be the sole source of O2 supply to tissue. However, the recent studies have demonstrated a longitudinal PO2 drop in arterioles, suggesting the possibility of O2 supply from arterioles. Furthermore, analysis of the diffusion process based on the PO2 drop has shown that the O2 diffusivity in the arteriolar wall was dramatically greater than in the tissue; thus, such a large O2 loss from arterioles seemed unlikely to be solely attributable to simple diffusion. To explain this discrepancy we hypothesized the O2 consumption by arteriolar walls was much higher than previously thought. In this study, we quantified the O2 consumption rate in arteriolar walls to evaluate its impact on the PO2 drop in the arterioles. Phosphorescence quenching microscopy was used to determine the intra- and perivascular PO2 values of rat cremaster arterioles both under normal condition and during vasodilation. Using the measured PO2 values, we calculated the O2 consumption rate of the arteriolar wall. Our results showed that 100 times more O2 is consumed by arteriolar walls, compared with in vitro vascular segments; consequently, O2 consumption by arteriolar walls could be the main cause of the PO2 drop in arterioles. Furthermore, we found the O2 consumption rate of the arteriolar walls under normal condition to be higher than during vasodilation and the O2 consumption to be dependent on the mechanical work of vascular smooth muscle. These findings suggested important roles of arterioles for O2 transport to tissue. Under resting skeletal muscle, to ensure blood supply to other organs with low systemic blood flow, the arterioles consume a large amount of O2 to restrict blood flow into skeletal muscle. While During exercise, arteriolar O2 consumption decreases as a result of vasodilation, thereby efficiently supplying O2 to the skeletal muscle of high O2 demand.