Human serum albumin is used to treat hypoproteinemia in neonates or used as a volume expander. We tested the hypothesis that the addition of human serum albumin to neonatal serum decreases the serum ionized calcium (iCa) concentration. Concentrated human serum albumin was added to 12 placental and cord serum samples to reach seven incremental concentrations from 0 to 20.0 g/L. Serum iCa concentration decreased significantly with the addition of serum albumin. From multiple regression analysis, the effect of albumin addition on serum iCa concentration had more marked effects at high baseline albumin or low baseline Ca concentrations. From this in vitro study, we speculate that fast infusion of albumin in human neonates has the potential for acutely lowering serum iCa concentration.