Protamine sulfate is the universal clinical antagonist to heparin and is used routinely after cardiovascular surgery to neutralize the anticoagulant function of heparin. Its clinical use, however, is associated with adverse effects including idiosyncratic fatal reactions. An examination of the mechanism of heparin neutralization and protamine toxicity suggests that the reversal of heparin anticoagulation may only require a small arginine-rich fragment of protamine to electrostatically dissociate antithrombin III from its binding to a specific pentasaccharide sequence in heparin. A review of literature indicates that chain-shortened peptide fragments derived from their parent proteins are normally accompanied with significantly reduced antigenicity and immunogenicity, which are two primary contributing factors to protamine-induced life-threatening toxic effects via an immunoglobulin-mediated pathway. Based on these observations, we propose our general hypothesis: if a chain-shortened low molecular weight protamine fragment containing the heparin-neutralizing domain could be derived directly from a native protamine, it could be a potent and nontoxic heparin antagonist. In this article, we present our experimental results to support the above hypothesis. LMWP fragments containing an intact arginine sequence and an average molecular weight of approximately 1.1 kDa were prepared successfully by enzymatic digestion of native protamine with thermolysin. In vitro studies demonstrated that such LMWP fragments completely neutralized the anticoagulant functions of heparin, based on the anti-Xa chromogenic assay and aPTT clotting time assay. Our in vivo results indicated that while administration of protamine to mice led to obvious production of antiprotamine antibodies, injection of LMWP did not elicit any detectable immunogenic responses. In addition, the LMWP fragments showed a significantly reduced antigenicity or, in other words, cross-reactivity towards the mice antiprotamine antibodies produced by the administration of protamine.