Cardiopulmonary bypass (CPB) procedures are frequently associated with massive inflammatory responses, resulting in a high rate of morbidity and mortality in routine cardiac operations. One recognized attribute of these deleterious responses is the synergic effect of heparin and protamine, which elicit the activation of the complement system in vivo. To circumvent such toxic effects following protamine reversal of heparin anticoagulation in the CPB procedures, we proposed that poly(ethylene glycol) (PEG)-modified protamine could retain the heparin-neutralization ability and yet diminish the induced complement activation by the formed heparin-protamine complexes (HPC), thereby providing highly improved pharmacological properties. PEGylation of protamine was carried out by utilizing N-hydroxysuccinimidyl (NHS) conjugation chemistry. Size exclusion chromatography (SEC), reverse-phase high performance liquid chromatography (RP-HPLC), and matrix-assisted laser desorption mass spectrometry (MALDI-MS) were used to assess the conjugation stiochiometry, the purity of the conjugates, and the site of PEG modification, respectively. The heparin-neutralizing activity was determined by using heparin affinity chromatography and various biological assays including the plasma-activated partial thromboplastin time (aPTT), anti-Xa, and anti-IIa methods. The potency in inducing complement activation was examined in vitro using the CH50 hemolytic assay. The PEG-modified protamine was successfully synthesized with a PEG/protamine stiochiometry of 1:1. Only one conjugation site for PEG that was located at the N-terminal end of protamine was obtained. In the biological evaluations, the PEG-modified protamine displayed a full retention of the heparin-neutralizing ability of protamine and a significantly reduced activity in complement activation following its complexation with heparin. Results from studies of the particle size and zeta potential indicated that the PEG-modified protamine formed substantially smaller aggregates with heparin, rendering them less effective in triggering the size-dependent complement responses. As with protamine, PEG-modified protamine exhibited an enhanced aqueous solubility, therefore attaining significantly improved pharmaceutical properties. These preliminary results suggested that the PEG-modified protamine conjugate might serve as a potential protamine substitute with improved therapeutic and pharmaceutical properties in heparin reversal.