Reducing the hemolobin (Hb)-O₂ binding affinity facilitates O₂ unloading from Hb, potentially increasing tissue mitochondrial O₂ availability. We hypothesized that a reduction of Hb-O₂ affinity would increase O₂ extraction when tissues are O₂ supply dependent, reducing the threshold of critical O₂ delivery (DO_2 CRIT). We investigated the effects of increased O₂ tension at which Hb is 50% saturated (P₅₀) on systemic O₂ uptake (O_2 SYS), DO_2 CRIT, lactate production, and acid-base balance during isovolemic hemodilution in conscious rats. After infusion of RSR13, an allosteric modifier of Hb, P₅₀ increased from 36.6 ± 0.3 to 48.3 ± 0.6 but remained unchanged at 35.4 ± 0.8 mmHg after saline (control, CON). Arterial O₂ saturations were equivalent between RSR13 and saline groups, but venous PO₂ was higher and venous O₂ saturation was lower after RSR13. Convective O₂ delivery progressively declined during hemodilution reaching the DO_2 CRIT at 3.4 ± 0.8 ml · min¹ · 100 g¹ (CON) and 3.6 ± 0.6 ml · min¹ · 100 g¹ (RSR13). At Hb of 8.1 g/l O_2 SYS started to decrease (CON: 1.9 ± 0.1; RSR13: 1.8 ± 0.2 ml · min¹ · 100 g¹) and fell to 0.8 ± 0.2 (CON) and 0.7 ± 0.2 ml · min¹ · 100 g¹ (RSR13). Arterial lactate was lower in RSR13-treated than in control animals when animals were O₂ supply dependent. The decrease in base excess, arterial pH, and bicarbonate during O₂ supply dependence was significantly less after RSR13 than after saline. These findings demonstrate that during O₂ supply dependence caused by severe anemia, reducing Hb-O₂ binding affinity does not affect O_2 SYS or DO_2 CRIT but appears to have beneficial effects on oxidative metabolism and acid base balance.