Arginine-chitosan conjugate (Arg-CS) was demonstrated to mediate significantly elevated expression of the transgenes in comparison to chitosan (CS). However, little is known about the hemocompatibility of Arg-CS/DNA nanoparticles (ACGN). This study evaluated the coagulation properties of the ACGN and CS/gene nanoparticles (CGN) by looking at the impact of the nanoparticles on the activated partial thrombin time (APTT), prothrombin time (PT), thrombin time (TT), and hemolysis ratio (HR). It demonstrated that both ACGN and CGN, at the concentration containing 50 μg/mL of DNA, led to moderate increases of APTT but no obvious change in PT and TT in vitro. Intravenous injection of CGN or ACGN (125 μg of DNA content/100 g of body weight) into rat did not result in any significant changes in PT but moderately increased APTT and TT. ACGN was demonstrated to be a slightly stronger inhibitor of blood coagulation as compared to CGN. No obvious hemolysis was induced by the nanoparticles. Taken together, the results in the present study indicated that introduction of arginine moieties into chitosan enhanced its anticoagulant property, which would be beneficial for applications in direct contact with blood.