To avoid bleeding complications, protamine is routinely used after cardiovascular surgery to neutralize the anticoagulant function of heparin. However, its clinical use is associated with adverse and sometimes fatal reactions. Based on literature review of the mechanism of heparin neutralization and protamine induced immunologic toxicity, we propose the following hypothesis: If a chain shortened low molecular weight protamine (LMWP) containing the heparin neutralizing domain could be derived from native protamine, it could be a potent and yet nontoxic heparin antagonist. In this study, we present results to validate this hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weight of approximately 1,100 daltons were successfully prepared by enzymatic digestion of protamine with thermolysin. In vitro studies show that such LMWP fragments completely neutralized the anticoagulant functions of heparin and LMWH, based on the anti-Xa chromogenic and aPTT clotting time assays. In vivo results reveal that although injection of protamine to mice led to obvious production of anti-protamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, these LMWP fragments exhibited a markedly reduced antigenicity and cross-reactivity toward the mice anti-protamine antibodies.