Erythrocytes participate in the matching of oxygen (O₂) delivery with local need in skeletal muscle via the release of O₂ and the vasodilator, ATP. It was reported that a concentration of insulin found in humans with insulin resistance inhibits low O₂-induced ATP release. However, in vivo, insulin is co-released with connecting peptide (C-peptide) at equimolar concentrations, but due to shorter insulin half-life, the peptides circulate at ratios of C-peptide to insulin ranging from 1:1 to 6:1. Here we investigate the hypothesis that C-peptide and insulin work synergistically to maintain low O₂-induced ATP release from human erythrocytes. Using a thin-film tonometer to alter O₂ tension we determined that either C-peptide or insulin alone inhibits low O₂-induced ATP release in a concentration-dependent manner; however, co-administration of the peptides at a 1:1 ratio does not (n=5, p<0.05). Since this ratio of C-peptide to insulin is not present in vivo for extended periods, we also investigated the effect of additional physiological ratios on ATP release. In the presence of insulin concentrations that would be found in fasting humans (0.05 nM), C-peptide to insulin ratios of 4:1 and 6:1 did not adversely affect low O₂-induced ATP release. However, at a concentration of insulin found in the peripheral circulation of humans under post-prandial conditions (0.5 nM), a ratio of C-peptide to insulin of 6:1 inhibited low O2-induced ATP release (n=5). These findings demonstrate a heretofore unrecognized synergism between C-peptide and insulin that could have physiological importance in the regulation of perfusion distribution in skeletal muscle.