Reducing the Hb-O₂ binding affinity facilitates O₂ unloading from hemoglobin (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 (DO2CRIT). We investigated the effects of increased P₅₀ on systemic O₂ uptake (VO2SYS), DO_2CRIT, 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 (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_2CRIT at 3.4±0.8 (CON) and 3.6±0.6 ml/minx100g (RSR13). At Hb of 8.1 g/L VO_2SYS started to decrease (CON:1.9±0.1; RSR13:1.8±0.2) and fell to 0.8±0.2 (CON) and 0.7±0.2 ml/minx100g (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 (O₂SD) was significantly less after RSR13 than after saline. These findings demonstrate that during O₂SD caused by severe anemia reducing Hb-O₂ binding affinity does not affect VO_2SYS or DO_2CRIT but appears to have beneficial effects on oxidative metabolism and acid base balance.