This is the first report on antithrombin effects of a phospholipase A 2 (RVAPLA 2 ) purified from venom of Daboia russelii russelii. The N-terminal sequence as well as in-gel tryptic digested peptides of RVAPLA 2 showed significant homology with PLA 2 s from Russell's viper venom. RVAPLA 2 demonstrated highest specific activity in hydrolyzing phosphatidylcholine (1.8 × 10 6 U/mg) with K m and V max values of 0.61 mM and 132.3 μmol/min, respectively. RVAPLA 2 exerted dose–dependent catalytic and strong anticoagulant activities; however, studies indicated dissociation of its catalytic and anticoagulant sites. The anticoagulant action of RVAPLA 2 was partially contributed by catalytic hydrolysis of plasma phospholipids. RVAPLA 2 showed strong anticoagulant effect via thrombin inhibition with a K i value of 380 nM as well as by binding to pro-coagulant phospholipids of plasma. In ex-vivo conditions, RVAPLA 2 (1.0 μM) was non-hemolytic and non-cytotoxic to mammalian cells. It did not inhibit the collagen-induced aggregation of platelets. RVAPLA 2 at a dose of 5 mg/kg was not lethal to mice after 48 h of injection. It demonstrated in vivo anticoagulant activity possibly due to targeting thrombin and binding with plasma phospholipids.